Human monoclonal antibodies against human cytokines and methods of making and using such antibodies

ABSTRACT

Human monoclonal antibodies against a human cytokine (such as a human interleukin, e.g., human IL-1α) and fragments of such antibodies are disclosed. Also disclosed are pharmaceutical compositions and methods employing the human monoclonal antibodies and fragments, methods for screening for human monoclonal antibodies against a human protein, methods for producing a cDNA library enriched in DNA encoding V H  and/or V L  chains of a human monoclonal antibody, cell lines for making the human monoclonal antibodies, and isolated DNA for making the human monoclonal antibodies and fragments of the invention.

The present application is the United States national applicationcorresponding to International Application No. PCT/US94/13188, filedNov. 21, 1994 and designating the United States, which PCT applicationis in turn a continuation-in-part application Ser. No. 93402846.5, filedNov. 23, 1993, the benefit of which applications are claimed pursuant tothe provisions of 35 U.S.C. 120, 363 and 365 (C).

FIELD OF THE INVENTION

The present invention relates to human monoclonal antibodies againsthuman cytokines and methods of making, identifying and using suchantibodies, preferably human monoclonal antibodies against humancytokines or lymphokines such as IL-1α, IL-1β, IL-4, IL-5, IL-6, IL-8,IL-10, TNF-α, etc.

BACKGROUND OF THE INVENTION

The applicability of human monoclonal antibodies (HuMAbs), especiallyHuMAbs to human cytokines, in therapy holds great promise; see, forexample, Griffiths et al., EMBO J., 12:725-734 (1993) and the review inLarrick et al., J. Biol. Response Modif., 5:379 (1986). However, theproduction of useful HuMAbs against human cytokines has proveddifficult.

Specifically, while the possible existence in human serum ofautoantibodies to human cytokines is mentioned in numerous articlesSuzuki et al., J. Immunol., 145:2140-2146 (1990) (IL-1α); Hansen et al.,Immunol. Letters, 30:133-140 (1991) (IL-1α); Bendtzen et al., Immunol.Today, 11:167-169 (1990) (IL-1α and TNF-α); Bendtzen et al., Immunol.Today, 10:222 (1989) (IL-1α and TNF-α); Saurat et al., J. Allergy Clin.Immunol., 88:244-256 (1991) (IL-1α); Suzuki et al., Clin. Exp. Immunol.,85:407-412 (1991) (IL-1α); Sunder-Plassmann et al., KidneyInternational, 40:787-791 (1991) (IL-1α); Gallay et al., Eur. CytokineNetw., 2:329-338 (1991) (IL-1α and IL-1β); Mae et al., LymphokineCytokine Res., 10:61-68 (1991) (IL-1α); Fomsgaard et al., Scand. J.Immunol., 30:219 (1989) (TNF-α); Hansen et al., Scand. J. Immunol., 33:777-781 (1991) (IL-6); Crabtree et al., Scand. J. Immunol., 37:65-70(1993) (IL-8); Bost et al., Immunology, 65:611-615 (1988) (IL-2); Rosset al., Clin. Exp. Immunol., 82:57-62 (1990) (IFN-α2b and IFN-γ); andCaruso et al., J. Immunol., 144:685-690 (1990) (IFN-γ)!, no one has beenable to produce an isolated and purified HuMAb to a human cytokine,especially a HuMAb having high affinity, e.g., a K_(a) of above about10⁹ M⁻¹. Some of the reasons are pointed out in the cited article byGriffiths et al. in EMBO. J.:

"Human monoclonal antibodies (mAbs) have huge potential for therapy, butare difficult to make by immortalizing B-lymphocytes. Furthermore, it isespecially difficult to generate human mAbs directed against humanantigens (anti-self antibodies), for example antibodies against solubleTNF to block septic shock, against membrane-bound carcinoembryonicantigen to image colorectal carcinoma, or against lymphocyte antigens todestroy tumour in lymphoma. This difficulty results from immunologicaltolerance mechanisms that prevent the antigen-driven expansion of B-cellclones with self specificities. After antibody gene rearrangement,virgin B-cells may display antibodies with self-reactivity, buttolerance mechanisms can lead to their deletion or to their anergy. Ithas been suggested that cells may be anergized if the antigen issoluble, but deleted if the antigen is membrane bound. B-cell tolerancedoes not seem to occur when concentrations of soluble antigen are low(in contrast to T-cell tolerance) and B-cells with poor affinities forantigen are not tolerized, even at higher antigen concentrations. Suchnon-tolerized B-cells are not usually expanded because they lack T-cellhelp, although proliferation can be induced artificially by usingpolyclonal B-cell activators.

It is estimated that 10-30% of B-lymphocytes in normal, healthyindividuals are engaged in making autoantibodies. However, the `naturalautoantibodies` produced do not lend themselves to therapeutic use asthey are often IgM, low affinity and polyreactive." (Citations omitted.)

Although the Griffiths et al. article speaks of "human self-antibodieswith high specificity," only single-chain V_(H) and V_(L) fragments areactually disclosed. Moreover, there is no disclosure in the article thatany of the heavy/light-chain combinations mentioned therein are actuallyfrom one human antibody. Moreover, the human antibody fragmentsdisclosed all have relatively low affinities, i.e., K_(a) s below 2×10⁷M⁻¹ and most below 10⁷ M-¹.

SUMMARY OF THE INVENTION

The present invention is directed to human monoclonal antibodies againsta human cytokine and to fragments of such antibodies having an affinityfor the cytokine of 10⁸ M⁻¹ or greater. The human monoclonal antibody(sometimes referred to herein as a HuMAb) or fragment preferably bindsto a human lymphokine, more preferably to a human interleukin, e.g.,human IL-1α, IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10, IL-11, IL-12, IL-13,especially IL-1α. The human monoclonal antibody or fragment of theinvention preferably has an affinity (K_(a)) to the human cytokine ofgreater than 10⁹ M⁻¹. The human monoclonal antibody or fragmentpreferably neutralizes the activity of the human cytokine. Humanmonoclonal antibodies of the IgG class are particularly preferred.

Another aspect of the invention involves a human monoclonal antibody ora fragment thereof comprising at least one CDR(complementarity-determining region) of an amino acid sequence definedby amino acids 1-122 of an amino acid sequence encoded by the nucleicacid sequence shown in SEQ ID NO. 1 and/or of an amino acid sequencedefined by amino acids 1-108 of amino acid sequence encoded by thenucleic acid sequence shown in SEQ ID NO. 2; or one or more somaticvariants of such sequences.

A preferred embodiment of the invention relates to a human monoclonalantibody or a human IL-1α binding fragment comprising:

a V_(H) segment having an amino acid sequence defined by amino acids1-122 of an amino acid sequence encoded by the nucleic acid sequenceshown in SEQ ID NO. 1 or by a CDR somatic variant thereof, and/or

a V_(L) segment having an amino acid sequence defined by amino acids1-108 of an amino acid sequence encoded by the nucleic acid sequenceshown in SEQ ID NO. 2 or by a CDR somatic variant thereof.

Preferably, the antibody comprises a V_(H) segment having an amino acidsequence defined by amino acids 1-122 of an amino acid sequence encodedby the nucleic acid sequence shown in SEQ ID NO. 1 and/or a V_(L)segment having an amino acid sequence defined by amino acids 1-108 of anamino acid sequence encoded by the nucleic acid sequence shown in SEQ IDNO. 2. More preferably, the antibody comprises V_(H) and V_(L) segmentshaving the amino acid sequences defined by amino acids 1-122 of an aminoacid sequence encoded by the nucleic acid sequence shown in SEQ ID NO. 1and by amino acids 1-108 of an amino acid sequence encoded by thenucleic acid sequence shown in SEQ ID NO. 2, respectively, or comprisesa CDR somatic variant of one or both of said amino acid sequences.Particularly preferred is an antibody having V_(H) and V_(L) segments ofthe amino acid sequences defined by amino acids 1-122 of an amino acidsequence encoded by the nucleic acid sequence shown in SEQ ID NO. 1 andamino acids 1-108 of an amino acid sequence encoded by the nucleic acidsequence shown in SEQ ID NO. 2, respectively, e.g., an antibody of thehuman IgG₄ isotype.

Preferred fragments of the invention comprise a V_(H) segment having anamino acid sequence defined by amino acids 1-122 of an amino acidsequence encoded by the nucleic acid sequence shown in SEQ ID NO. 1and/or a V_(L) segment having an amino acid sequence defined by aminoacids 1-108 of an amino acid sequence encoded by the nucleic acidsequence shown in SEQ ID NO. 2, e.g., a Fv, single-chain Fv, Fab orF(ab')₂ fragment. Preferably, the IL-1α binding fragment of theinvention has an affinity of 10⁷ M⁻¹ or greater, more preferably of 10⁸M⁻¹ or greater.

Another aspect of the invention involves isolated nucleic acids (DNAS)which encode a human monoclonal antibody or fragment in accordance withthe present invention. Preferably, the isolated nucleic acid comprises:

a nucleotide sequence defined by base numbers 58-423 of SEQ ID NO. 1 orby a CDR encoding somatic variant thereof, or a functional equivalent ofsuch a nucleotide sequence, and/or

a nucleotide sequence defined by base numbers 67-390 of SEQ ID NO. 2 orby a CDR encoding somatic variant thereof; or

a functional equivalent of one or both of said nucleotide sequences.

In a preferred embodiment, the isolated nucleic acid comprises anucleotide sequence defined by base numbers 58-423 of SEQ ID NO. 1and/or base numbers 67-390 of SEQ ID NO. 2.

Still other aspects of the invention relate to a pharmaceuticalcomposition comprising at least one human monoclonal antibody orfragment in accordance with the invention and a pharmaceuticallyacceptable carrier, and to the use of an anti-IL-1α HuMAb or IL-1αbinding fragment of the invention to treat inflammation.

The invention also includes a method for screening a solution for adesired human monoclonal antibody against a human protein comprising

(1) contacting the solution with labeled protein and polyclonal ormonoclonal anti-human Ig (i.e., anti-IgA, IgD, IgE, IgG and/or IgM)coupled to a substrate or with labeled protein and protein G coupled toa substrate; and

(2) determining if a desired human monoclonal antibody is present in thesolution by detecting labeled protein in any immunoprecipitated product.

Preferably, the solution is a collection of supernatants from a human Bcell mixture. In a preferred embodiment, the solution is screened usingeither polyclonal or monoclonal anti-human Ig coupled to a substrate orwith protein G coupled to a substrate. These screening methods can beused to prepare and identify a purified mixture of human B cells or asingle human B cell clone by the steps of

serially diluting a human B cell mixture giving a positive result in thescreen to provide a purified mixture of human B cells or single B cellclones; culturing said purified mixture of human B cells or single humanB cell clones; and

screening supernatants from said cultured purified mixture of human Bcells or single B cell clones by the above methods to determine if thedesired human monoclonal antibody is present in the supernatants of saidcultured purified mixture of human B cells or single B cell clones.

Still another aspect of the invention involves a method of producing acDNA library enriched in DNA encoding a V_(H) and/or V_(L) chain of ahuman monoclonal antibody against a desired antigen comprising the stepsof:

producing a CD40-crosslinked and EBV-transformed, immortalized and/oractivated B cell population containing immortalized and/or activated Bcells expressing said human monoclonal antibody;

cloning subpopulations of said immortalized and/or activated B cellpopulation and identifying a subpopulation which contains immortalizedand/or activated B cells expressing said human monoclonal antibody;

preparing a cDNA library using the mRNA from said subpopulation tocreate a repertoire of DNAs encoding at least the V_(H) and/or V_(L)chain of the human monoclonal antibodies expressed by said subpopulationof immortalized and/or activated B cells.

Preferably, this method further comprises:

identifying DNA within said library that encodes at least the V_(H)and/or V_(L) chain of the desired human monoclonal antibody; and

using said DNA to produce a human monoclonal antibody against thedesired antigen or an antigen-binding fragment of such an antibody.

In one such embodiment, a population or subpopulation which containsimmortalized and/or activated B cells expressing said human monoclonalantibody is identified by the screening method described above.

Preferably, the repertoire of DNAs is incorporated into vectors capableof displaying said V_(H) and/or V_(L) chain on the surface of a hostcell, host cells are transformed with said vectors, and host cells thatdisplay a V_(H) and/or V_(L) chain that binds to the desired antigen areidentified by affinity binding to the desired antigen. The DNAs thatencode said V_(H) and V_(L) chains that bind to the desired antigen canthen be operatively linked to DNA encoding any necessary constant-regionchains for a human immunoglobulin so as to create a DNA sequenceencoding a heavy chain of a human monoclonal antibody against thedesired antigen and a DNA sequence encoding a light chain of a humanmonoclonal antibody against the desired antigen.

Other important aspects of the invention include (1) a human B cell lineestablished by EBV-transformation and CD40-crosslinking, whichestablished cell line (preferably an antibody-producing clone) producesa human monoclonal antibody against a human cytokine, and (2) a processfor making a human monoclonal antibody against a human cytokinecomprising the steps of

establishing an immortalized and/or activated human B cell populationfrom a patient having antibodies that bind to the human cytokine, saidimmortalization and/or activation comprising infecting the B cells withEpstein-Barr virus and crosslinking the CD40 of such B cells;

culturing said immortalized and/or activated B cells;

isolating multiple clones from such immortalized and/or activated Bcells, each of which clones secretes a human monoclonal antibody thatbinds to the cytokine; and

using one or more of such clones to produce one or more human monoclonalantibody or a fragment thereof.

In this process nucleic acid encoding the human monoclonal antibody orfragment is preferably used to produce the desired antibody or fragment.Alternatively, the clone produced in the process is hybridized with amyeloma or heteromyeloma cell to produce a hybridoma that proliferatesin culture and produces the desired antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical representation showing the amount of bound ¹²⁵I-IL-1α (cpm) versus the concentration of IL-1α (pM) in the assay "X3Affinity for Human IL-1α" described below.

FIG. 2 is a graphical representation showing the amount of ¹²⁵ I-IL-1αbound on EL4 cells (cpm) versus the concentration of HuMAb X3 (μg/ml) inthe assay "Inhibition of Human IL-1α Receptor Binding" described below.

FIG. 3 is a graphical representation showing the amount of ¹²⁵ I-IL-1αprecipitated (cpm) versus the concentration of HuMAb X3 (μg/ml) in theassay "Cross-Reactivity of the Human Monoclonal Antibody X3" describedbelow using excess human IL-1β, human IL-1Ra or human IL-1α to protectagainst immunoprecipitation by HuMAb X3.

FIG. 4A is a graphical representation showing the EL4/CTLLproliferation-- ³ H!TdR uptake (cpm) versus the concentration of IL-1α(pg/ml) in the assay "Inhibition of Human IL-1α-induced IL-2 Secretionby EL4 Cells" described below.

FIG. 4B is a graphical representation showing the EL4/CTLL proliferation(% of response) in the presence of human IL-1β or human IL-1α versus theconcentration of HuMAb X3 (μg/ml) in the assay "Inhibition of HumanIL-1α-induced IL-2 Secretion by EL4 Cells" described below.

FIG. 5A is a graphical representation showing the IL-6 production(ng/ml) in the presence of either 0.1 μg/ml or μg/ml of HuMAb X3 versusthe concentration of IL-1α (pg/ml) in the assay "Inhibition of HumanIL-1α-induced IL-6 Production by Human Synoviocytes" described below.

FIG. 5B is a graphical representation showing the IL-6 production (% ofresponse) in the presence of either human IL-1β or IL-1α versus theconcentration of HuMAb X3 (μg/ml) in the assay "Inhibition of HumanIL-1α-induced IL-6 Production by Human Synoviocytes" described below.

FIGS. 6A and 6B are graphical representations showing the EL4/CTLLproliferation (cpm×10⁻³) in the presence of rabbit anti-IL-1β, rabbitanti-IL-1α, or two concentrations of HuMAb X3 versus the amount ofparaformaldehyde-fixed (PFA-fixed) Monocytes per well without and withLPS stimulation, respectively, in the assay "Inhibition of MembraneAssociated Human IL-1α Activity" described below.

FIGS. 7A, 7B, 7C and 7D are graphical representations showing theproduction of IL-6 (ng/ml) in the presence of CTL IgG₄ /κ, IL-1Ra orHuMAb X3 versus the amount of Monocytes/well, PFA-monocytes/well,LPS-Monocytes/well and PFA/LPS-Monocytes/well, respectively, in theassay "Inhibition of IL-6 production in cocultures of synoviocytes andmonocytes" described below.

FIG. 8A is a graphical representation showing the amount of ¹²⁵ I-IL-1α(cpm) precipitated in the assay "Standard Immunoprecipitation Protocolwith Protein G" described below with various antibody materials,including the natural HuMAb X3 and the recombinant light and heavychains from the HuMAb X3.

FIG. 8B is a graphical representation showing the amount of ¹²⁵ I-IL-1α(cpm) precipitated in the assay "Cross-Reactivity of the HumanMonoclonal Antibody X3" described below using excess human IL-1β, humanIL-1Ra and human IL-1α to protect against immunoprecipitation by naturalHuMAb X3 or recombinant HuMAb X3.

FIG. 9A is a graphical representation showing EL4/CTLL proliferation-- ³H!TdR uptake(cpm) versus the concentration of purified natural HuMAb X3(μg/ml) in the assay "Inhibition of Human IL-1α-induced IL-2 Secretionby EL4 cells" described below.

FIG. 9B is a graphical representation showing IL-6 production (ng/ml)versus the concentration of purified natural HuMAb X3 or theconcentration of purified recombinant HuMAb X3 (μg/ml) in the assay"Inhibition of Human IL-1α-induced IL-6 production by HumanSynoviocytes" described below.

DETAILED DESCRIPTION OF THE INVENTION

The invention may employ a B cell population including resting B cellswhich retain their surface bound immunoglobulin and/or activated B cellswhich secrete HuMAbs. If desired, the B cell population may be sorted toselect for activated B cells or for resting B cells, e.g., as describedbelow and in WO 91/09115.

A starting human B cell population for use in providing a humananti-cytokine HuMAb (or a subsequence thereof that binds to thecytokine) in accordance with the present invention can be identified bymeans conventional in the art, e.g., by the methods described in thearticles listed in the Section "Background of the Invention" above. Asmall amount of blood can be taken from patients and tested for Igagainst the desired cytokine, e.g., by ELISA, radioimmunoprecipitationassay, western blotting, etc. Patients who react positively are sourcesof B cells that can be used to immortalize and isolate a clone producingthe desired HuMAb as described further below. A larger sample forcloning can then be taken from each patient identified by the aboveprocedures.

Suitable sources of B cells from a selected patient include peripheralblood, tonsils, adenoid tissue, spleen (in the case of removal foranother medical necessity) or any other source of B cells from the body.Typically, peripheral blood is employed as the B cell source.

The blood is first treated to separate the peripheral blood lymphocytes(PBLs) from the red blood cells and platelets by means conventional inthe art. For example, the peripheral blood may be diluted with anappropriate isotonic medium, e.g., RPMI 1640 medium (cat. 041-01870 M.Gibco, USA). The diluted blood is loaded onto a suitable separationmedium such as FICOLL™ (available from Pharmacia, Sweden). Aftercentrifugation, the PBLs may be aspirated from the interface between theplasma and the FICOLL™. The purified PBLs may be frozen in liquidnitrogen for later use. The plasma is then analyzed by conventionaltechniques such as radioimmunoprecipitation assay, ELISA, westernblotting, etc., to confirm the presence of significant amounts of thedesired antibody (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA, IgD, IgM and/or IgEantibody) against the cytokine of interest.

The purified PBLs may be used directly or may be further enriched and/orsorted as discussed below. For example, T-cells may be removed byresetting with 2-aminoethylisothiouroniumbromide-treated sheeperythrocytes. Further selection for an antigen-specific B cellsubpopulation can be carried out by a variety of techniques includingpanning, immunoadsorbent affinity chromatography, fluorescent-activatedcell sorting (FACS), etc. These techniques are described for example inCasali et al., Science, 234:476-479 (1986); U.S. Pat. No. 4,325,706; andMage, Hubbard et al., Parks et al. and Haegert, in Meth. Enzymol.,108:118-124, 139-147, 197-241 and 386-392 (1984), respectively. The PBLsmay also be treated with magnetic beads whose surface is coated with amaterial to selectively sort the desired B cells. Such beads may becoated, e.g., with anti-Ig isotype for the desired Ig to be separated,with anti-surface antigen to select for non-naive B cells, or with apurified cytokine.

The resulting enriched and/or sorted B cell population is then subjectedto the B-cell immortalization and/or activation process described in WO91/09115. Briefly, the B cells are transformed with Epstein-Barr virus(EBV) and their CD40 molecules are crosslinked. Reference is made to WO91/09115 for the variations that may be employed in thisactivation/immortalization process.

The treated B cell population may be washed by an appropriate isotonicmedium (e.g., with RPMI 1640), pelleted and then resuspended in medium.The cells are then transformed with EBV by the addition of a suitableEBV strain, preferably a strain such as the one released by the B95.8cell line available from the ATCC (ATCC CRL 1612). The amount of EBVused may vary depending on the strain of the virus and the number of Bcells to be transformed. For example, with a sample containing 14×10⁶non-sorted PBLs, 200 μl of a suspension of a concentrated EBV (strainB95.8) is typically used. Incubation with the virus is typically carriedout for about 1 to 24 hours, preferably for about 2 hours, at 37° C.;but other conditions may be employed, if desired.

The EBV-infected cells are preferably washed and resuspended in anappropriate enriched medium such as Yssel's modified Iscove's medium 15%Fetal Calf Serum (FCS) Yssel et al., J. Immunol. Methods, 72:219-227(1984)!. The concentration of the PBLs in the suspension may varydepending, for example, on whether a sorting step for antigen-specific Bcells was performed as described above. Lower concentrations can beemployed when PBLs have been enriched in the desired B cells. Typicalconcentrations for non-selected PBLs are from about 1×10³ to about 5×10⁴cells/ml. If the B cell population is first sorted as discussed above,the concentration may be decreased depending upon the efficiency of thesorting, e.g., up to about 1×10² cells/ml.

An agent capable of crosslinking CD40 antigen is added to the suspendedcells. The crosslinking agent may include T-cells, other transfectedcells expressing CD40 ligand, or membranes therefrom. Other suitableagents are described in WO 91/09115. Preferably, the agent is animmobilized monoclonal antibody specific for the CD40 antigen, e.g.,immobilized on irradiated fibroblasts expressing the human or murineFc-gamma receptor (ATCC CRL 10680).

The monoclonal antibody to CD40 can be any which binds to the CD40marker on the B cells of the suspension and also to the Fc-gammareceptor of the L-cells. Preferably, the monoclonal antibody is selectedfrom MAb 89 and G28-5. These antibodies are described in Valle et al.,Eur. J. Immunol., 19:1463-1467 (1989) and Ledbetter et al., J. Immunol.,138:788-794 (1987), respectively. The hybridoma corresponding to MAb 89has been deposited with the European Collection of Animal Cell Cultures,PHLS Center for Applied Microbiology and Research, Porton Down,Salisbury Wilts. SP4 OJG, U.K. under accession No. 89091401.

Typically, the PBLs, the CD40 antibody and the L-cells are simply mixedtogether in appropriate amounts. The CD40 antibody may be present in aconcentration of from about 0.01 μg/ ml to about 50 μg/ml, preferablyfrom about 0.1 μg/ml to about 5 μg/ml, more preferably about 0.5 μg/ml.

The treated cell suspension is divided among an appropriate number ofwells of a tissue microplate to provide a suitable cell concentrationfor amplification and screening. If enriched suspensions are employed asthe result of an antigen-selective screening as discussed above, fewercells per well may be used.

Typically, the initial culture phase takes 10-20 days in the case ofnon-selected PBLs and 5 days or even less in the case of anantigen-specific enriched B cell population, which would allow anearlier detection of specific antibodies. During this phase, freshmedium is added as necessary. The duration of this initial culture phaseis adjusted to allow detection of the antigen-specific B cells, whilepreventing them from being overgrown by non-specific B cells. A sampleof supernatant from each well is screened by an appropriate assay forthe desired HuMAb positive characteristics, e.g., byradioimmunoprecipitation assay, ELISA, western blotting, etc.

In a preferred screening method, supernatants are contacted with alabeled protein (e.g., radiolabeled with ¹²⁵ I) and polyclonal ormonoclonal anti-human Ig coupled to a substrate or insoluble support.The anti-human Ig can be a mixture of isotypes (i.e., IgG₁, IgG₂, IgG₃,IgG₄, IgA, IgD, IgE and/or IgM) or an individual isotype (e.g., IgG₄).Alternatively, if one is screening for an IgG HuMAb, the supernatantscan be contacted with a labeled protein and protein G coupled to asubstrate or insoluble support. The presence of the desired HuMAb isdetermined by detecting labeled protein in the immunoprecipitatedproduct. The immunoprecipitation screens (with anti-human Ig and withprotein G) may be employed serially.

Cell lines which test positive for the desired HuMAb characteristics arecloned (3-10 cells/well) and subcloned (0.5-1.0 cells/well) bytechniques well-known in the art, e.g., by culturing in limitingdilution conditions for 7-20 days in additional medium as needed.Supernatants of the clones are screened by the procedures describedabove.

Positive clones are expanded in a larger volume and amplified byconventional incubation.

HuMAb can be purified from the supernatant of the amplified clones byconventional immunoglobulin-purification methodology. For example, theHuMAb may be precipitated with solid ammonium sulfate, reconstituted insterile water, and dialyzed extensively against a buffer such asphosphate-buffered saline (PBS). The dialysate may then be applied to animmunoaffinity column, e.g., a column having anti-human Ig or Protein Gcovalently coupled to Sepharose. After washing, the desired HuMAb may beeluted from the column by any appropriate eluent, e.g., acidic buffer,chaotropic agents, etc. for example, see Current Protocols inImmunology, edited by John E. Coligan et al., John Wiley and Sons, NewYork!.

By the term "human monoclonal antibody" as used herein, we mean toinclude HuMAbs that are isolated from human B cells as discussed above(e.g., whether the antibody is prepared by culturing the immortalizedand/or activated human B cells or recombinantly from human B cell cDNAsencoding such a HuMAb and whether or not the antibody is bound to amolecule which can alter its biological activity, e.g., a receptor orligand, an enzyme, a toxin, a carrier, etc.) and antibodies that aremade by recombining the variable portions of a HuMAb of the presentinvention of one isotype (e.g., an IgG₄) with the constant region of ahuman antibody of another isotype (e.g., a human IgG₁, IgG₂, IgG₃, IgG₄,IgA, IgD, IgM or IgE). Recombinant methods for making these HuMAbs aredescribed below.

By the terms "fragment" or "subsequence" of a HuMAb of the presentinvention, we mean an antibody fragment such as an Fab, F(ab')₂, Fv,single-chain binding protein, or any other binding polypeptide whichcontains one or more complementarity determining regions (CDRs) of thevariable region of a light or heavy chain of a HuMAb of the presentinvention (e.g., an Fab, Fv, CDR, etc. of a HuMAb in accordance with thepresent invention either alone or linked to any desired molecule whichcan alter its biological activity, e.g., a receptor or ligand, anenzyme, a toxin, a carrier, etc.). These fragments can be prepared bywell-known methods. For example, fragments can be made from thefull-length HuMAb protein, e.g., by papain or pepsin cleavage, or bychemical oxidation, followed by separation of the resulting fragments.Alternatively, recombinant DNA technology may be used. For example, cDNAencoding the variable regions of both heavy and light chains may beengineered to produce the Fv portion of the HuMAb of the invention. See,for example, the methodology of U.S. Pat. No. 4,642,334 which may beemployed.

By the terms "CDR somatic variant" and "CDR encoding somatic variant" asused herein we mean an amino acid or nucleic acid sequence correspondingto SEQ ID NO. 1 and/or SEQ ID NO. 2 or a subsequence of SEQ ID NO. 1and/or SEQ ID NO. 2 containing at least one CDR or CDR-encoding regionthereof, but having at least one mutation, addition and/or deletion inone or more of the CDRs or CDR-encoding region of the sequence orsubsequence, such that an anti-IL-1α human monoclonal antibody includingsaid at least one mutation, addition and/or deletion has an IL-1αbinding affinity of 10⁸ M⁻¹ or greater, preferably 10⁹ M⁻¹ or greater.

By the term "affinity" as used herein, we mean the measure of thebinding strength between an antigenic determinant and an antigen bindingsite of a human monoclonal antibody of the invention or a fragmentthereof as measured by the affinity constant (Ka), e.g., by the methoddescribed below.

By the term "V_(H) segment" as used herein, we mean the variable regionof the heavy chain of a human monoclonal antibody of the invention.

By the term "V_(L) segment" as used herein, we mean the variable regionof the light chain of a human monoclonal antibody of the invention.

By the term "Fv fragment" as used herein, we mean an antigen bindingfragment of an antibody that contains the variable regions of the heavy(V_(H)) and light (V_(L)) chains. Those V_(H) and V_(L) regions can belinked to form a single-chain Fv (scFv).

By the term "Fab fragment" as used herein, we mean the antigen bindingfragment resulting from the digestion with papain of a human monoclonalantibody of the invention.

By the term "F(ab')₂ fragment" as used herein, we mean the antigenbinding fragment resulting from the digestion with pepsin of a humanmonoclonal antibody of the invention.

By the term "functional equivalent" as used herein, we mean a nucleicacid sequence that encodes the same amino acid sequence as theidentified nucleic acid sequence.

The terms "activated" B cell and "activation" of a B cell as used hereinindicate a human B cell that has been CD40 crosslinked andEBV-transformed and expresses and secretes human antibodies.

The B cell clones of the invention may be used in conventional DNArecombinant methods to produce the HuMAbs of the invention or fragmentsthereof. For example, RNA from the B cell clones may be isolatedaccording to the single-step method described by (Chomczynski et al.,Anal. Biochem., 162 156-9 (1987). Briefly, about 10⁷ cells are lysed inguanidinium thiocyanate denaturing solution. After acidification of themixture with 2M sodium acetate, pH4, RNA is extracted with phenol andchloroform/3-methyl-1-butanol. RNA is then precipitated withisopropanol, the RNA pellet is redissolved in denaturing solution,reprecipitated with isopropanol, and washed with 75% ethanol.

cDNA is obtained by reverse transcription, e.g., using the SuperscriptReverse Transcriptase Kit (cat. 20898 BRL, Gaithersburg, Md., USA), witholigo dT₁₂₋₁₈ primers (Cat. 27.7858-01, Pharmacia, Uppsala, Sweden). ThecDNA is then used as template in a polymerase chain reaction (PCR). Theprimers may be designed to include restriction sites, to allow for thedirectional cloning of the PCR products. For the heavy chain, primersspecific for the leader sequence of all the different human V_(H)families are used individually in conjunction with primers located atthe 3'-end of the constant region corresponding to the isotypepreviously determined by isotyping the HuMAb by ELISA or otherappropriate method (e.g., radioimmunoprecipitation assay, etc.). Thelight chain is amplified with individual combinations of primerscorresponding to the 3'-end of the kappa or lambda chain in conjunctionwith a series of primers annealing to the leader sequence of the V kappaor the V lambda genes. Thus, full-length heavy and light chains startingat the initiation codon in the leader sequence and ending at the stopcodon may be generated.

After appropriate restriction cleavage, both full-length heavy chainsand lull-length light chains can then be cloned in any appropriateexpression vector designed to be compatible with the restricted PCRproducts. Appropriate vectors include for example baculovirus vectorsand plasmids compatible with CHO cells or other host cells. Examples ofsuitable vectors and hosts are described in the review "Engineeredantibody molecules" in Immunol. Reviews, 130 (1992). Heavy and lightchains can be cloned individually in distinct vectors, or in tandem inone vector. The recombinant plasmids or viral vectors may be cloned inbacteria, and a few clones may be sequenced on both strands to check forthe absence of alteration of the insert. One clone each for the heavychain and for the light chain, or one clone containing both chains, maythen be selected for expression in the appropriate host cells. Dependingon the vector used, it will be introduced into appropriate prokaryoticor eukaryotic cells either by transfection or by infection. The cellsexpressing the recombinant HuMAb are cloned, supernatant fluid from thecultured cells is collected, and the HuMAb therein can be purified, e.g.by immunoaffinity, HPLC or any other appropriate methods.

The full-length PCR product for the heavy chain can be modified forexample to replace the original heavy-chain constant region by anotherone, so replacing one isotype by another, e.g., replacing a human IgG₄isotype by a human IgG₁, IgG₂, IgG₃, IgA, IgD, IgM or IgE isotype. Thiscan be accomplished through a second PCR using as 5'-primer the V_(H)leader specific primer, and as 3'-primer a primer annealing to the3'-end of the V_(H) region (e.g., IgG₄) and including a tailcorresponding to the 5'-end of the desired constant region (e.g., IgG₁,IgG₂, etc.) cloned in an appropriate plasmid. After amplification, theDNA generated encoding the heavy chain may be digested with appropriateenzymes and ligated into the new expression vector, which will nowcontain the sequence of the desired heavy chain. This will allow theproduction of a recombinant HuMAb with the variable region of theoriginally isolated HuMAb (e.g., the variable region from an IgG₄ HuMAb)and the constant region of a different human isotype (e.g., the constantregion of a human IgG₁, IgG₂, etc.). This type of recombinant HuMAb willhave the characteristic binding of the IgG₄ HuMAb, but will be able todisplay the effector functions normally associated with the human IgG₁,IgG₂, etc. isotype. The same method can be used to replace an isotypeother than IgG₄ by another different isotype.

Hybridomas may also be made with the B cells of the invention bytechniques conventional in the art. For example, the B cells of theinvention may be fused with an appropriate myeloma cell or with aheterohybridoma cell to increase or stabilize the immunoglobulinsecretion; see for example Kudo et al., J. Immunol. Methods, 145:119-125(1991); Zanella et al., J. Immunol. Methods, 156:205-215 (1992); andDarveau et al., J. Immunol. Methods, 159: 139-143 (1993).

The HuMAbs and fragments of the present invention may be usedtherapeutically to treat existing symptoms associated with the antigenof interest. For example, IL-1α, IL-1β and TNF-α are identified asinflammatory cytokines and thus HuMAbs to such cytokines or fragments ofsuch HuMAbs may be useful for treating inflammation including chronic oracute inflammatory reactions such as rheumatoid arthritis,osteoarthritis, inflammation associated with asthma, inflammatory boweldisease, regulating fever associated with inflammation, pain relief ininflammation, etc. For example, bowel diseases may benefit from atreatment with an anti-IL-1α HuMAb of the invention, e.g., HuMAb X3,since anti-IL-1α antibodies abolished the crypt hyperplasia in thejejunum of mice suffering from graft-versus-host disease enteropathy:see Mowat et al., Immunology, 80:110-115 (1993). Also, since IL-1α hasbeen suspected to play a role in psoriasis see Romero et al., J. Invest.Dermatol., 93:518-522 (1989)!, an anti-IL-1α HuMAb of the invention maybe useful in treatment of psoriasis. Allergy may be another target foran anti-IL-1α HuMAb of the invention at both the regulatory and effectorlevels. At a regulatory level, IL-1 has been shown to be involved in thedifferentiation of naive T lymphocytes into TH₂ T cells. At the effectorlevel, clinical trials with soluble IL-1 receptor have shown a strikinginhibition of wheal and flare reaction in allergen-challenged allergicpatients. Furthermore, the anti IL-1 HuMAbs and anti IL-1-bindingfragments of the present invention are antagonists to IL-1 and thereforewill be useful for the same indication as other known IL-1 antagonists,which either are in clinical trials for or have been shown in theliterature to be useful in models of septic (endotoxin) shock,experimental autoimmune encephalomyelitis, cerebral malaria,graft-versus-host disease and chronic myelogenous leukemia see, forreview, Dinarello, Immunol. Reviews, 127:119-146 (1992); and Dinarelloet al., N. Engl. J. Med., 328:106-113 (1993)!. Finally, IL-1α has beenreported to act as an autocrine growth stimulator for human thyroid andgastric carcinoma cells Ito et al., Cancer Res., 53:4102-4106 (1993)!,as well as adult T cell leukemias Shirakawa et al., Cancer Res.,49:1143-1147 (1989)!. Thus, an anti-IL-1α HuMAb of the invention may beuseful in the treatment of tumors.

Further examples of the utilities of other antagonists of and/orantibodies to other cytokines are described in Henderson et al., TiPS,13:145-152 (1992) and in Mire-Sluis, TIBTECH, 11:74-77 (1993). TheHuMAbs and fragments of the present invention may also be usedprophylactically to prevent or inhibit the occurrence of such symptomsassociated with the antigen of interest.

The HuMAbs of the invention may be particularly useful, e.g., intreating chronic diseases, in view of the long half-lives of HuMAbs(e.g., about 21 days Adair, Immunol. Reviews, 130:5-40 (1992)!) comparedto other cytokine antagonists (about 30 minutes for IL-1 receptorantagonist Granowitz et al., Cytokine, 4:353-360 (1992)!). This longerhalf life may allow a bimonthly or monthly administration of the HuMAb.

The HuMAb or fragment thereof of the present invention may be used aloneor in combination with at least one other HuMAb or fragment to form acocktail or with another antiinflammatory drug. For example, such acocktail may include two or more of the HuMAbs of the invention, each ofwhich binds to one or more epitopes on a cytokine of interest. When sucha cocktail is employed, the proportions of the various HuMAbs orfragments may vary depending, for example, upon their bindingcharacteristics.

The HuMAbs and fragments of the present invention are preferablyadministered in the form of a pharmaceutical composition containing atherapeutically or prophylactically effective amount of at least onesuch HuMAb or fragment in combination with a pharmaceutically acceptablecarrier. Any appropriate carrier may be employed, i.e., a compatible,non-toxic material suitable for delivery of the HuMAb or fragment in thedesired dosage form, e.g., oral, parenteral (subcutaneous, intramuscularor intravenous), or topical dosage forms. Suitable carriers includesterile water, sterile buffered water, sterile saline, etc. Specialpharmaceutical compositions to insure a sustained release of the HuMAband/or fragment may also be employed.

The concentration of the HuMAb or fragment of the invention in thepharmaceutical compositions may vary, e.g., from about 0.1 μg/ml toabout 1 mg/ml, preferably from about 1 μg/ml to about 100 μg/ml. Theconcentration used will depend upon the number of HuMAbs and/orfragments thereof employed in the composition, their bindingcharacteristics and the dosage form selected. The dose will be adjustedin a conventional manner by the skilled artisan to levels appropriate toachieve the desired result in vivo.

As mentioned above, the HuMAbs and/or fragments of the invention can beused prophylactically or therapeutically. Thus, the agent may beadministered before the onset of symptoms or after the symptoms haveappeared. The HuMAbs and/or fragments of the invention will beadministered in a dose effective to provide the desired alleviation ofsymptoms. Amounts effective for this purpose will depend upon manyfactors, e.g., the severity of the symptoms in the patient.

The HuMAb or fragment of the invention may be administered in dosages offrom about 0.001 μg/kg to about 1 mg/kg, e.g., about 0.01 μg/kg to about1 μg/kg, preferably from about 0.01 μg/kg to about 0.1 μg/kg. The properdosage of a HuMAb or fragment of the invention for a particularsituation will be determined by using common practices in the art.Generally, treatment may be initiated with smaller dosages that are lessthan the optimum dose of the agent. Thereafter, the dosage may beincreased by small amounts until the optimum effect under thecircumstances is reached. The amount and frequency of administration ofthe HuMAb or fragment of the invention will be regulated according tothe judgment of the attending clinician considering such factors as age,condition and size of the patient as well as severity of the symptomsbeing treated.

The HuMAbs and fragments of the invention may also be used fordiagnostic purposes in the same manner as other antibodies and fragmentsare currently used in the art. For example, the HuMAbs and fragments ofthe invention can be used in assays for the cytokine to which they bindor in an immunopurification procedure to isolate an antigen to whichthey bind. The HuMAbs and fragments may be used either labeled (e.g.,with a radioisotope, fluorescent group, enzyme or other appropriateligand) or unlabeled, as is conventional in the art for the particularassay of interest (e.g., in a sandwich assay with a second labeledantibody). The HuMAbs and fragments may be used in agglutination assays,enzyme immunoassays, etc. They could for example be used to calibrate adosage of cytokine-specific IgG in the serum or in any other biologicalfluid. Thus, the labeled or unlabeled forms of the HuMAbs and fragmentsof the invention may be employed as elements of kits for purposes ofperforming the desired assay.

The invention disclosed herein is illustrated by the following Examples,which should not be construed to limit the scope of the disclosure.Alternative methods within the scope of the invention will be apparentto those skilled in the art.

Unless otherwise indicated, percentages for solids in solid mixtures,liquids in liquids, and solids in liquids are weight/weight,volume/volume and weight/volume, respectively.

EXAMPLES General Methods and Reagents

Recombinant human IL-1 alpha (IL-1α) and recombinant human IL-1 Receptorantagonist (IL-1Ra) were expressed in E. coli by standard methods andpurified by ion-exchange and gel chromatographies.

Recombinant human IL-1 beta (IL-1β) was purchased from Genzyme (Boston,Mass.).

¹²⁵ I-labeled recombinant human IL-1α (specific activity: 1200-2200Ci/mmole) was from Du Pont De Nemours (Wilmington, Del.).

Mouse monoclonal antibody to human IL-1α and rabbit neutralizingantibodies against human IL-1α and human IL-1β were from Genzyme(Cambridge, Mass.).

Protein G (from Group C Streptococcus sp.) coupled to Sepharose 4B™, andanti-human Ig polyvalent immunoserum (IgG fraction) coupled to agarosewere obtained from Sigma Chemical Co. (St Louis, Mo.).

Tissue culture media, fetal calf serum (FCS), L-glutamine, Hepes buffer,and Phosphate Buffered Saline (PBS) were from GIBCO (Paisley, UK).Bovine serum albumin (BSA) was from Sigma Chemical Co. and gentamycinfrom Schering-Plough (Levallois-Perret, France).

The murine thymoma cell line EL4 (ATCC, TIB 181) was maintained in RPMI1640 supplemented with 10% FCS, 2 mM L-glutamine, 50 μg/ml gentamycinand 5×10⁻⁵ M 2-mercaptoethanol (2-ME) in a humidified 37° C. chamberwith 5% CO₂.

The murine IL-2-dependent cytotoxic T cell line (CTLL-2) (ATCC, TIB 214)was maintained in RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine,50 μg/ml gentamycin, 5×10⁻⁵ M 2-ME and 20 U/ml recombinant human IL-2 ina humidified 37° C. chamber with 5% CO₂.

Human synoviocytes were isolated from rheumatoid synovial biopsiesobtained from rheumatoid arthritis patients undergoing knee or wristsynovectomy, or joint replacement as described in Dechanet et al., J.Immunol., 151:4908-4917 (1993). Fat and fibrous tissues were removed.The resulting fragments of synovium were finely minced into small piecesand digested with 4 mg/ml collagenase (Worthington, Freehold, N.J.) inPBS for 2-3 hours at 37° C. After centrifugation, cells were resuspendedin α-MEM (Gibco) medium Minimum Essential Medium Eagle! supplementedwith 2 mM L-glutamine, 20 mM Hepes buffer, 10% FCS and 50 μg/mlgentamycin. Cells were cultured in 100 mm culture Petri dishes, inhumidified 5% CO₂ atmosphere. After adherence for 48 hours, non-adherentcells were removed and adherent cells were cultured until confluence.Then cells were cultured in 150 cm² culture flasks after trypsintreatment. Synoviocytes were used from passage 3 to passage 8. They werenegative for the expression of CD1, CD2, CD3, CD19, CD14 and HLA-DR asdetermined by flow cytometry analysis on a FACScan (Becton Dickinson,Sunnyvale, Calif.) after staining with specificfluorescein-isothiocyanate (FITC) conjugated monoclonal antibodies(mAbs) (Becton Dickinson, Mountain View, Calif.).

The transformant Epstein-Barr virus (EBV), strain B 95.8, was producedby culturing transformed marmoset leukocytes (ATCC, CRL 1612)essentially as described by Miller and Lipman Proc. Natl. Acad. Sci.USA, 70:190 (1973)!.

The ltk⁻ transfected mouse fibroblastic L cell line (ATCC CRL 10680)stably expressing the human Fcγ receptor II (FcγRII or CDw32), and themouse anti-human CD40 monoclonal antibody, mAb 89, were obtained asdescribed in WO 91/09115.

Standard Immunoprecipitation Protocol with Protein G

In order to identify the presence of human antibodies (IgG) againsthuman IL-1α in biological samples (sera, plasma, etc.) or culturesupernatants, an immunoprecipitation assay was carried out usingradio-labeled recombinant human IL-1α and protein G-Sepharose asprecipitating reagent. This assay allowed the identification of the foursub-classes of human IgG (IgG₁, IgG₂, IgG₃ and IgG₄). Typically, 50 μlof sera/plasma from patients or 50 μl of culture supernatants (both usedat appropriate dilution in PBS, 1% BSA) were incubated for 45 minutes atroom temperature with 50 μl (50 pM) of human ¹²⁵ I-IL-1α (diluted in PBS1% BSA) in a well of a 96-well filtration microplate MultiScreen-HA™(Millipore Co., Bedford, Mass.) whose bottom was composed of anitrocellulose membrane (HATF 0.45 μm). Each sample was tested induplicate. Then, 50 μl of a dilution of protein G coupled to Sepharose4B™ (Sigma Chemical Co) (15 ml beads diluted to 50 ml in PBS 1% BSA)were added to each well and incubated for 45 minutes at roomtemperature. The wells were then washed three times with PBS using avacuum manifold (Millipore Co.), and the dried membranes were collectedinto appropriate vials using a special collector system (Millipore Co.).The radioactivity corresponding to the complexes ¹²⁵ I-IL-1α/anti-IL-1αwas counted in a Wizard gamma-counter (Wallac Oy, Turku, Finland).Positive and negative controls were performed for each plate, using arabbit anti-human IL-1α antiserum (Genzyme, Cambridge, Mass.) or anunrelated antiserum respectively. Specificity of the human ¹²⁵ I-IL-1αprecipitation obtained with the tested samples was further confirmedthrough its inhibition by preincubation of those samples with a 100-foldexcess of unlabeled recombinant human IL-1α.

Derived lmmunoprecipitation Protocols

The above standard immunoprecipitation assay has been modified in orderto identify human antibodies to human IL-1α of isotypes other than IgGor to better identify the IgG subclass and light chain of suchantibodies contained in patient biological fluids, e.g., sera or culturesupernatants. The principle of the different assays was the same as inthe standard protocol, but the precipitating reagent, e.g. proteinG-Sepharose, was changed. The following reagents were used: agarosebeads coupled with goat polyspecific antibodies to human IgM, IgG andIgA (Sigma Chemical Co); Affi-Gel 10™ gel (Bio-Rad laboratories,Richmond, Calif.) coupled, according to the manufacturer's instructions,with specific goat antibodies to human IgA heavy chain, human lambdalight chain or human kappa light chain (Sigma Chemical Co.) or coupledwith mouse mono-clonal antibodies to human IgG, heavy chain, human IgG₂heavy chain, human IgG₃ heavy chain or human IgG₄ heavy chain(Calbiochem Co., La Jolla, Calif.

These protocols may be employed with other antigens of interest bysubstituting an appropriately labeled antigen for the ¹²⁵ I-IL-1α in theassays.

Detection of Human Antibodies to Human IL-1α in Human Biological Fluids

Detection of naturally occurring autoantibodies to human IL-1α inbiologiical fluids, e.g., sera or plasma, was performed by using theradioimmuno-precipitation assay described above. Blood samples fromhealthy donors or sick patients (particularly patients suffering fromautoimmune diseases, infectious diseases or neurologic disorders) werescreened. 10% of the samples from healthy donors (101/1009) containedIgG antibodies to human IL-1α, in that, when diluted 1:10, theysignificantly precipitated ¹²⁵ I-labeled human recombinant IL-1α withprotein G-Sepharose. Some samples also contained IgA autoantibodies tohuman IL-1α, as determined by immunoprecipitation of ¹²⁵ I-labeled IL-1αwith appropriate anti-human IgA reagents coupled to beads. An increasedfrequency (15.9%, 59/370) of IgG anti-IL-1α autoantibodies was observedin sera of patients with autoimmune diseases. Precipitation ofradio-labeled human IL-1α was specific since it was completely inhibitedby pre-incubation of the positive samples with a 100-fold excess ofunlabeled human IL-1α.

Autoantibodies to human IL-1α were titrated using theimmunoprecipitation assay for serial dilutions of positive sera orplasma. Then, sera or plasma samples with high titers of anti-IL-1αantibodies were also tested for their ability to inhibit the binding ofhuman ¹²⁵ I-IL-1α to its receptors expressed on the murine thymoma EL4cells. Serial dilutions (in RPMI 1640, 1% BSA, 20 mM Hepes) of positivesera or plasma were pre-incubated for 1 hour at 4° C. with a fixedconcentration (70 pM) of human ¹²⁵ I-IL-1α, in a final volume of 100 μl.Experiments were performed in conical 1 ml Eppendorf tubes or inV-bottomed microtiter plates (Nunc, Roskilde, Denmark), and each samplewas tested in triplicate. Then 1×10⁶ (100 μl) of EL4 cells in RPMI 1640,1% BSA, 20 mM Hepes were added to each tested point and the mixures wereincubated for 3 hours at 4° C. Cells were then washed and centrifugedthree times at 4° C., and the radioactivity corresponding to cell-bound¹²⁵ I-IL-1α was counted in a Wizard gamma-counter (Wallac). Non-specificbinding was measured in the presence of a 100-fold excess of unlabeledhuman IL-1α. Most of the sera or plasma containing autoantibodies tohuman IL-1α was found to block human IL-1α binding to its receptor.Patients were selected according to these criteria. Examples of thereactivity of 5 different sera are shown in Table 1 below.

                  TABLE 1    ______________________________________    Detection of IgG autoantibodies to human IL-1α in human sera            .sup.125 I-IL-1α precipitated                        .sup.125 I-IL-1α bound to EL4 cells                  with Protein G        (%    Sera  Dilution                  (cpm)         cpm     inhibition)    ______________________________________    none           787          4516    negative          1/5     1082          4490    C     1/5     3860          903     (80)          1/10    3844          2365    (48)          1/20    2554          3233    (28)    S     1/5     3697          1685    (63)          1/10    2763          3156    (30)          1/20    2061          3864    (14)    T     1/5     3392          2332    (48)          1/10    2692          2945    (35)          1/20    1953          3559    (21)    V     1/5     4130          642     (86)          1/10    3741          655     (85)          1/20    2486          1976    (56)    X     1/5     3866          701     (84)          1/10    3723          2122    (53)          1/20    2593          2666    (41)    ______________________________________     C, S, T, V, and X designate serum samples from healthy persons and     patients.

In a similar manner, autoantibodies against other cytokines in humanbiological fluids, such as sera, plasma, etc., are detected bysubstituting, for example, ¹²⁵ I-TNF-α, ¹²⁵ I-IL-1β, ¹²⁵ I-IL-6 or ¹²⁵I-IL-10 for ¹²⁵ I-IL-1α in the above procedure. Samples from suchpatients are then EBV-transformed, CD40-activated, screened andamplified by the methods described below to produce subpopulationsand/or single clones of B cells producing HuMAbs against IL-1β, TNF-α,IL-6 or IL-10. Detection of IgG autoantibodies to human IL-10 in a humanserum is shown in Table 2 below.

                  TABLE 2    ______________________________________    Detection of IgG autoantibodies to human IL-10 in human serum                  .sup.125 I-IL-10 precipitated                                .sup.125 I-IL-10 precipitated after                  with Protein G                                protection with unlabeled    Sera  Dilution                  (cpm)         IL-10 (cpm)    ______________________________________    none          405           375    negative          1/2     430           410    positive          1/2     1425          450          1/4     1142          417          1/8     927           391          1/16    734           397          1/32    442           401    ______________________________________

Generation of X3, a Human Monoclonal Antibody to Human IL-1α

Plasma from a selected patient (identified as X) was found toprecipitate human ¹²⁵ I-IL-1α with protein G and inhibited the bindingof human ¹²⁵ I-IL-1αto EL4 cells in the protocols described above (seeTable 1 above).

EBV Transformation and CD40 Activation of B Lymphocytes from Patient X

40 ml of peripheral blood collected on EDTA treated tubes were obtainedfrom patient X. Blood was diluted 1:1 with PBS and loaded onto a Ficoll™(Pharmacia, Uppsala, Sweden) density gradient. Peripheral bloodmononuclear cells (PBMNC) were collected at the interface aftercentrifugation (30 min, 600 g). Cells were washed four times in PBS, and39×10⁶ PBMNC were finally obtained, with a cell viability superior to95%, as estimated by Trypan Blue dye exclusion.

The cells were pelleted and resuspended in 1 ml of RPMI complete mediumwhich consisted of RPMI 1640 supplemented with 10% heat-inactivated FCS,2 mM L-glutamine and 50 μg/ml gentamycin. Then, 500 μl of a 100×concentrated Epstein-Barr virus (EBV) suspension (strain B95.8) wereadded, and this mixture was incubated for 2 hours at 37° C. under 5% CO₂in a humidified incubator. The cells were then washed once in RPMIcomplete medium and the pellet was resuspended at 5×10⁴ cells/ml inYssel's modified Iscove's medium Yssel et al., J. Immunol. Methods72:219-227 (1984)! supplemented with 15% heat-inactivated FCS, 2 mML-glutamine, 50 μg/ml gentamycin. Irradiated L cells (7,000 rads) stablyexpressing the FcγRII (CDw32) were added at a final concentration of5×10⁴ cells/ml, together with the murine monoclonal anti-human CD40antibody mAb 89 used at a final concentration of 0.5 μg/ml. 100 μlaliquots of this mixture were then distributed in each well ofround-bottomed 96-well culture plates (Nunc), and the plates wereincubated at 37° C. under 5% CO₂ atmosphere in humidified incubator.After 5 days of incubation, 125 μl of fresh culture medium containing0.5 μg/ml anti-CD40 mAb 89 were added to each well.

Screening of Culture Supernatants

After 10 days of incubation, the culture supernatants were screened forthe presence of human anti-IL-1α antibodies. Thus, 60 μl of culturemedium were collected from each well, and the 12 different supernatantscorresponding to the 12 wells of each line of the culture plates werepooled in one microtube. This operation was facilitated by the use of aBIOMEK 1000 work station (Beckman Instruments, Fullerton, Calif.). Then,50 μl of the pooled supernatants were screened individually for thepresence of antibodies that bind human IL-1α by immunoprecipitation ofrecombinant human ¹²⁵ I-IL-1α with polyspecific anti-human IgM, IgA andIgG antibodies coupled to agarose (Sigma Chemical Co). A total of 13positive pools were identified with this screening assay. A positiveresult was immediately confirmed by immunoprecipitation of human ¹²⁵I-IL-1α with protein G coupled to Sepharose, indicating the presence ofhuman IgG antibodies to human IL-1α.

The 13 pools were then identified and split. At day 11, 50 μl of culturesupernatants were harvested from each well corresponding to the 13pools, and they were tested individually by immunoprecipitation of human¹²⁵ I-IL-1α with protein G. A total of 13 different positive wells werethus identified. They were designed X1 to X13.

Characterization of the Human Antibodies to Human IL-1α from the 13Positive Cell Lines

The 13 initial positive cell lines (X1 to X13) were expanded in order toproduce supernatants for further analysis, and the cells were frozen andstored in liquid nitrogen. The positive results of the 13 differentlines were verified at different times by immunoprecipitation of human¹²⁵ I-IL-1α with protein G.

Experiments performed with positive culture supernatants have shown thatthe 13 cell lines secreted human antibodies that inhibit the binding ofhuman ¹²⁵ I-IL-α on EL4 cells.

The isotype of the human anti-IL-1α antibodies contained in thesesupernatants was determined by using an immunoprecipitation assay ofhuman ¹²⁵ I-IL-1α with Affi-Gel 10 beads coated with specific antibodiesagainst human IgG₁, IgG₂, IgG₃ or IgG₄ heavy chain, or antibodies tohuman kappa (κ) or lambda (λ) light chain. Of the human anti-IL-1αantibodies secreted by the 13 cell lines, three were of IgG₁ /κ isotype,one was of IgG₄ /λ isotype, and the nine others were of IgG₄ /κ isotype.In particular, the human anti-IL-1α antibody X3 was of IgG₄ /κ isotype.

In a manner similar to the above methods, B cells from 14 other patientsthat tested positive in the detection screen described above were alsoactivated with CD40 and transformed with EBV, screened and expanded.PBLs isolated from these 14 selected patients were submitted to EBVinfection, cultured in the CD40 system and screened as described abovefor patient X. 28 other B cell lines secreting anti-IL-1α antibodieswere identified. Thus, among a total of 482×10⁶ PBLs used (from 15different patients), 41 cell lines secreting anti-human IL-1α antibodieswere identified; 40 secreted IgG and one secreted IgA antibodies tohuman IL-1α, and all these 41 cell lines precipitated ¹²⁵ I-IL-1α andinhibited its binding on EL4 cells. Details are shown in Table 3 below.Whereas the 40 positive wells other than the one from patient X asdescribed above lost anti-IL-1α precipitating activity upon subsequentcloning and subcloning, the B cells in positive wells may be used toidentify and isolate other anti-IL-1α HuMAbs by the other methodsdescribed below, e.g., by repertoire cloning.

                  TABLE 3    ______________________________________    Generation of anti-IL-1α secreting human B cell lines          No. PBLs PBLs/well                            No. positive                                    Anti-IL-1α                                            Binding    Patient          (× 10.sup.6)                   (× 10.sup.3)                            cell lines                                    Isotype Inhibition    ______________________________________    C     50       5        9       IgG     +    D     45       5        1       IgG     +    F     21       5        0       --      -    H     40       5        1       IgG     +    I     41       5        1       IgG     +    P     90       5        12      IgG + IgA                                            +    Q     20       5        0       --      -    R     15       4        1       IgG     +    S     40       1        0       --      -    T     10       1        0       --      -    U     16       1        0       --      -    V     15       1        0       --      -    W     15       1        2       IgG     +    X     39       5        13      IgG     +    Y     25       5        1       IgG     +    TOTAL 482               41      40 IgG  +                                    + 1 IgA +    ______________________________________

Cloning of the 13 Positive Cell Lines from Patient X

Twelve days after the initiation of the culture, the 13 positive initialcell lines (X1 to X13) were cloned by limiting dilution at 5 cells/wellin 96-microwell plates (round-bottomed). Aliquots of cells wereharvested, enumerated and resuspended at 50 cells/ml in complete culturemedium containing 5×10⁴ /ml irradiated (7,000 rads) CDw32 transfected Lcells and 0.5 μg/ml anti-CD40 mAb 89. 100 μl of this suspension wasdistributed in each well and culture plates were incubated at 37° C., 5%CO₂. After 6 days of incubation, 125 μl of fresh Yssel's modifiedIscove's medium were added to each well. Between 10 and 24 days afterthe cloning initiation, 50 μl of supernatant were harvested from thewells showing a cell growth, and screened individually for anti-IL-1αantibodies by the immunoprecipitation assay performed with anti-humanIgM, IgA and IgG antibodies coupled with agarose or protein G-Sepharose.The cell line X3 gave rise to three positive clones: X3A, X3B and X3C.

Subcloning of X3A, X3B and X3C

The three positive clones X3A, X3B and X3C were then subcloned at 1cell/well in complete culture medium without feeder cells. The screeningwas performed at different days after the initiation of the culture, byimmunoprecipitation assay as described above. A total of 261EBV-transformed cell lines secreting human anti-IL-1α antibodies wereobtained. The cells were expanded in RPMI complete medium. Culturesupernatants were frozen and stored at -20° C. Cells were frozen at1×10⁶ to 5×10⁶ cells/ml and kept in liquid nitrogen.

Antibodies Isotyping in Conditioned Media

Three subclones: X3A-16G5, X3B-14G10 and X3C-20G10, obtained asdescribed above, were selected and maintained in culture in RPMI 1640supplemented with 10% FCS, 2 mM L-glutamine and 50 μg/ml gentamycin.Conditioned media from these three clones were collected and tested(dilution 1:1 in PBS, 0.05% Tween-20) in enzyme-linked immunosorbentassays (ELISA) specific for human IgM, IgG or IgA isotypes, and forhuman IgG₁, IgG₂, IgG₃ or IgG₄ subclasses.

Only a human IgG₄ immunoglobulin has been identified in conditionedmedia from the three selected subclones X3A-16G5, X3B-14G10 andX3C-20G10, thus suggesting the monoclonality of these threeEBV-transformed cell lines. Furthermore, sensitive PCR analysis ofV_(H), C_(H), V_(L), and C_(L) usage indicated the presence of a uniqueIg transcript in the isolated B cell clones. The subclone X3A-16G5 wasselected for subsequent analysis and the human monoclonal antibodyproduced by it will be referred to hereinafter as X3.

Purification of the Human Monoclonal Antibody X3

The subclone X3A-16G5 was used to produce large quantities of humanmonoclonal antibodies to human IL-1α. This clone was stable for morethan 5 months, and was continuously amplified in RPMI 1640 mediumsupplemented with 10% FCS, 2 mM L-glutamine and 50 μg/ml gentamycin toallow the generation of large number of cells. Then cells werecollected, washed twice in PBS to remove FCS protein contamination, andrecultured for 5 days at the initial concentration of 5×10⁵ cells/ml inRPMI 1640 supplemented with 2 mM L-glutamine, 50 μg/ml gentamycin and1×Nutridoma-HU (Boeringer Mannheim GmbH, Mannheim, Germany). Under theseconditions, this clone produced 3 to 5 mg/l of monoclonal IgG₄.

Conditioned culture medium was then collected, filtered andconcentrated. Immunoglobulins were then precipitated with 2.4M (NH₄)₂SO₄. After centrifugation, the precipitate was dissolved in TPP buffer(20 mM H₃ PO₄, pH 7) and loaded to an affinity column of proteinG-Sepharose 4B (Sigma Chemical Co.) previously equilibrated with TPPbuffer. The column was then washed once with TPP buffer, 1M NaCl, andthree times with TPP buffer alone. The human antibody X3 was eluted fromthe column with 0.1M glycine buffer, 0.4M NaCl, pH 2.7. The pH wasimmediately adjusted to pH 8 by addition of TRIZMA Base 1M, pH 12, andthe purified antibody was dialyzed against PBS.

The quality of the X3 purifications was verified by subjecting theobtained preparations to polyacrylamide gel electrophoresis (SDS-PAGE)in a 10% gel under reducing conditions essentially as described byLaemmli Nature 227:680-685 (1970)!, and after subjecting the gel tosilver staining.

To verify that the human antibody X3 was not denatured during thepurification, purified preparations of X3 were tested for their abilityto precipitate human ¹²⁵ I-IL-1α in the standard assay described aboveusing protein G-Sepha rose.

X3 Affinity for Human IL-1α

To determine the affinity of the human monoclonal antibody X3 for humanIL-1α, the equilibrium association constant of X3/IL-1α complexes wasmeasured. Purified human antibody X3 was incubated with increasingconcentrations of human ¹²⁵ I-IL-1α (10 to 500 pM) in a final volume of250 μl in RPMI 1640, 1% BSA, 20 mM Hepes. Each tested condition wasperformed in triplicate. Nonspecific binding controls were performed induplicate by addition of 50 nM unlabeled recombinant human IL-1α. After4 hours' incubation at 4° C., 200 μl of each sample were distributed inone well of 96-well special titration plates (MultiScreen-HA, 0.45 μm)containing 50 μl of protein G coupled to Sepharose. After 1 hour'sincubation at 4° C., plates were washed four times with PBS and driedmembranes were collected from each well. Radioactivity corresponding tothe complexes ¹²⁵ I-IL-1α/anti-IL-1α/protein G-beads retained on themembranes was counted using a Wizard gamma-counter (Wallac). Specificbinding of human ¹²⁵ I-IL-1α was calculated, then plotted versus freehuman ¹²⁵ I-IL-1α concentrations and subjected to Scatchard analysisusing a Ligand software (FIG. 1).

The value of the equilibrium affinity constant (K_(a)) obtained for thehuman monoclonal antibody X3 was 5×10⁹ M⁻¹.

Inhibition of Human IL-1α Receptor Binding

The ability of human monoclonal antibody X3 to inhibit the binding ofradiolabeled human IL-1α to IL-1 receptors expressed on murine thymomaEL4 cells was investigated using both conditioned medium and thepurified antibody. Serial dilutions (in RPMI 1640, 1% BSA, 20 mM Hepes)of positive culture supernatants or purified HuMAb X3 were pre-incubatedfor 1 hour at 4° C. with a fixed concentration (70 pM) of human ¹²⁵I-IL-1l, in a final volume of 100 μl. Experiments were performed inconical 1 ml Eppendorf tubes or in V-bottomed microtiter plates (Nunc,Roskilde, Denmark), and each sample was tested in triplicate. Then,1×10⁶ (100 μl) of EL4 cells in RPMI 1640, 1% BSA, 20 mM Hepes were addedto each tested point and incubated for 3 hours at 4° C., then washedthree times, and the radioactivity corresponding to cell-bound ¹²⁵I-IL-1α was counted in a Wizard gamma-counter (Wallac). Non-specificbinding was measured in the presence of a 100-fold excess of unlabeledhuman IL-1α.

The results presented in FIG. 2 indicated that the antibody X3 blocks ina dose-dependent manner the binding of radiolabeled human IL-1α on EL4cells. The concentration of antibody X3 required to block 50% ofreceptor binding (IC₅₀) was found to be 0.015 μg/ml (100 pM) for aconstant concentration of 70 pM of radiolabeled human IL-1α.

Cross-Reactivity of the Human Monoclonal Antibody X3

To determine whether antibody X3 specifically binds to human IL-1α,different preparations of IL-1 were tested for their ability to protectthe precipitation of human ¹²⁵ I-IL-1α by the human antibody X3.Different dilutions (in PBS, 1% BSA) of purified antibody X3 werepreincubated for 1 hour at room temperature without or with an excess(10 nM) of either recombinant human IL-1α (as positive control),recombinant human IL-1β or recombinant human IL-1 receptor antagonist(IL-1Ra). Then human ¹²⁵ I-IL-1α (50 pM) was added to each sample andreaction was incubated for 45 minutes at room temperature. Each samplewas tested in duplicate in MultiScreen-HA™ plates. Precipitation of ¹²⁵I-IL-1α/anti-IL-1α complexes was done with protein G coupled toSepharose and radioactivity was counted in a Wallac Wizardgamma-counter.

The results as shown in FIG. 3 showed that neither an excess ofunlabeled human IL-1β nor an excess of unlabeled human IL-1Ra protectedthe immunoprecipitation of human ¹²⁵ I-IL-1α by antibody X3, whileunlabeled human IL-1α completely inhibited radiolabeled human IL-1αprecipitation. These results indicate that the human monoclonal antibodyX3 specifically recognized human IL-1α, but not human IL-1β and humanIL-1Ra.

Inhibition of Human IL-1α-induced IL-2 Secretion by EL4 Cells

The biological activity of human IL-1 was measured by its ability tostimulate IL-2 production by the murine thymoma subline EL4-6.1 Zubleret al., J. Immunol., 134:3662-3668 (1985)!. The IL-2 production wasfurther determined using the CTLL-2 assay Gillis et al., J. Immunol.,120:2027-2033 (1978)!. The proliferation of IL-2-dependent CTLL cells isproportional to the concentration of IL-2 produced by EL4 cells in thefirst step of culture.

Different concentrations of the purified human monoclonal antibody X3were incubated for 30 minutes at 37° C. with various concentrations ofrecombinant human IL-1α or human IL-1β in a final volume of 100 μl/wellin flat-bottomed 96-well culture plates (Falcon, Oxnard, Calif.). Eachexperimental point was done in triplicate and reagent dilutions wereperformed in culture medium composed of RPMI 1640 supplemented with 2 mML-glutamine, 10% heat-inactivated FCS, 50 μg/ml gentamycin and 5×10⁻⁵ M2-ME. Then, 100 μl of a suspension of EL4 cells (5×10⁵ cells/ml) inculture medium containing 0.2 μg/ml ionomycin (Sigma Chemical Co) wereadded to each well. After 24 hours' incubation at 37° C. under 5% CO₂ ina humidified incubator, cell-free supernatants were harvested and testedfor their IL-2 concentrations.

Aliquots of 50 μl of these supernatants were distributed inflat-bottomed microtiter plates and incubated with 50 μl/well of asuspension of IL-2-dependent CTLL cells (5×10⁴ cells/ml) which had beenwashed twice prior to the assay to remove contamination of IL-2 added tomaintain their continuous growth. After 36 hours' incubation (37° C., 5%CO₂), cells were pulsed for 4 hours with 0.5 μCi/well ³ H-Thymidine(specific activity: 25 Ci/mmol, CEA, Saclay, France). ³ H-Thymidineuptake was then measured by standard techniques after harvesting cellson glass fiber filters with a BETAPLATE™ 96-well harvester (Pharmacia)and counting in a BETAPLATE™ liquid scintillation counter (Wallac).Results were expressed in cpm±standard deviation of culture triplicatesand are shown in FIG. 4.

The human monoclonal antibody X3 specifically inhibits humanIL-1α-induced IL-2 production by EL4 cells, but not the IL-2 secretioninduced by human IL-1β. The concentration of antibody X3 required toblock 50% of IL-2 secretion induced by 50 pg/ml (2.8 pM) human IL-1α(IC₅₀) was found to be 0.1 μg/ml (700 pM).

Inhibition of Human IL-1α-induced IL-6 Production by Human Synoviocytes

Human synoviocytes were isolated from rheumatoid synovial biopsiesobtained from rheumatoid arthritis patients.

Different concentrations of the human monoclonal antibody X3 wereincubated for 30 minutes at 37° C. with various concentrations ofrecombinant human IL-1α or human IL-1β in a final volume of 100 μl/wellin flat-bottomed microtiter plates (Falcon). Each experimental point wasdone in triplicate and reagent dilutions were performed in culturemedium composed of α-MEM (Gibco) supplemented with 2 mM L-glutamine, 10%heat-inactivated FCS, 50 μg/ml gentamycin and 20 mM Hepes. Then, 100μl/well of a suspension of human synoviocytes (5×10⁴ cells/ml in culturemedium described above) were added. After 7 days of incubation (37° C.,5% CO₂) supernatants were harvested and tested for their human IL-6concentrations using a specific ELISA.

Results shown in FIG. 5 indicate that the human monoclonal antibody X3specifically inhibits human IL-1α-induced IL-6 production by humansynoviocytes, but not the IL-6 secretion induced by human IL-1β. With aconstant concentration of 50 pg/ml (2.8 pM) human IL-1α, the IC₅₀ wasfound to be 0.02 μg/ml (150 pM) of human monoclonal antibody X3.

Inhibition of Native Human IL-1α Biological Activity

All the experiments described above were performed using a recombinantform of human IL-1α. In order to demonstrate that the human antibody X3also neutralizes the biological activity of native human IL-1α, wetested the ability of the antibody X3 to inhibit IL-1-related activitiescontained in conditioned media or lysates of human mononuclear cellsstimulated with lipopolysaccharide (LPS).

To produce native IL-1, PBMNC were isolated from healthy donors bycentrifugation on standard Ficoll™ density gradient. PBMNC were washedand 10⁷ cells were then incubated at 37° C. under 5% CO₂ in one well ofa 6-well tissue-culture plate (Falcon) in 2.5 ml complete culture mediumwhich consisted of RPMI 1640 supplemented with 2 mM L-glutamine, 10% FCSand 50 μg/ml gentamycin. After 1 hour's incubation, non-adherent cellswere removed and the adherent cells were washed three times with culturemedium maintained at 37° C. Then adherent cells were re-cultured for 24hours in 2.5 ml complete culture medium containing 1 μg/ml LPS (E. coli,serotype 0111:B4) (Sigma Chemical Co.). After this incubation period,supernatant corresponding to the conditioned medium was harvested andcentrifuged to remove contaminating cells. Adherent cells were washedthree times with cold PBS and removed by treatment at 4° C. with EDTA0.02% (Sigma) and gentle scrapping with a rubber policeman. Cells werethen washed twice, centrifuged, resuspended in 500 μl culture medium andlysed by successive freezings in liquid nitrogen. The cell lysate wasthen centrifuged (10,000×g, 15 min, 4° C.) and the upper phase wascollected and adjusted to 1 ml with complete culture medium.

The ability of the human monoclonal antibody X3 to block the nativehuman IL-1 activity contained in conditioned medium or lysate fromLPS-stimulated adherent human mononuclear cells was investigated usingthe EL4/CTLL assay, because such conditioned medium and lysate maycontain significant amounts of LPS and IL-6 which may interact in thesynoviocyte assay, but not in the EL4/CTLL assay.

Serial dilutions of conditioned medium or lysate from LPS-stimulatedadherent cells were preincubated for 30 minutes without or with 1 μg/mlpurified antibody X3, 1 μg/ml non-related IgG₄ /κ human monoclonalantibody (as negative control) or 1 μg/ml rabbit neutralizing antibodiesagainst human IL-1α (Genzyme) (as positive control). Each condition wasdone in triplicate, in 96-well flat-bottomed culture plates under afinal volume of 100 μl/well. Then 100 μl/well of EL4 cells (5×10⁵cells/ml) were added and incubated 24 hours at 37° C. under 5% CO₂ inhumidified incubator. Culture supernatants were then collected and theirIL-2 concentrations were determined using the CTLL assay.

Results presented in Table 4 below show that the human antibody X3inhibited the IL-1 activity contained in lysate but not in conditionedmedium obtained from LPS-stimulated human mononuclear cells. Theneutralizing rabbit anti-human IL-1α antiserum shared the sameactivities as antibody X3, while the unrelated human antibody did notinhibit the IL-1 activity neither in conditioned medium, nor in celllysate. These results indicate that the antibody X3 recognizes andneutralizes human native IL-1α (which is mostly present in the cytosoland associated with the membrane of LPS-stimulated monocytes), but notnative IL-1β (which is principally secreted after LPS-stimulation).

                  TABLE 4    ______________________________________    Inhibition of native human IL-1 by antibody X3                  EL4/CTLL proliferation    LPS-stimulated                   .sup.3 H!-Thymidine uptake (cpm × 10.sup.-3)    adherent MNC           Control        Rabbit    Preparation             Dilution Medium   IgG.sub.4 /κ                                      X3    anti-IL-1α    ______________________________________    Lysate   1/40     84.4     115.4  18.2  22.2             1/400    26.8     35.7   2.9   4.2    Supernatant             1/400    85.8     98.0   82.9  89.4             1/4000   5.5      4.9    5.1   4.2    ______________________________________

The reactivity of HuMAb X3 with Cynomolgus IL-1 was also tested. Monkeyblood mononuclear cells were isolated, stimulated with LPS and lysedafter 24 hours' incubation. Increasing concentrations of lysate inducedEL-4 cells to secrete IL-2, and this activity was inhibited by thepolyclonal rabbit anti-human IL-1α. HuMAb X3 was also able to block themonkey IL-1α, but its activity appeared to be lower than that observedwith human IL-1α.

Inhibition of Membrane-Associated Human IL-1α Activity

The reactivity of HuMAb X3 with membrane-associated IL-1α was studied byusing highly purified human monocytes metabolically inactivated afterparaformaldehyde (PFA) fixation. Monocytes were isolated from peripheralblood by elutrial centrifugation essentially as described by De Mulderet al., J. Immunol. Methods, 47:31-38 (1981). Purity of the differentfractions obtained was assessed by flow cytometry analysis, andpreparations containing more than 90% CD14 positive cells were selected.Monocytes were then cultured with or without LPS (1 μg/ml) in Tefloncell culture bags for 24 hours at 37° C., 5% CO₂. Then cells were washedtwice in PBS and resuspended in PBS containing 1% PFA (Sigma) for 10minutes at 20° C. Cells were then washed three times with glycine buffer(150 mM glycine, 75 mM NaCl, pH 7.4) and three times with RPMI 1640complete culture medium. Serial dilutions of PFA-fixed monocytes werethen incubated 30 minutes at 37° C. in a final volume of 100μl/well withor without either 1 or 10 μg/ml monoclonal antibody X3, or 10 μg/mlrabbit anti-IL-1α or anti-IL-1β antiserum. Cultures were performed inflat-bottomed 96-well culture plates. Then 100 μl of EL4 suspension(5×10⁵ cells/ml) were added to each well. After 48 hour's incubation,IL-2 secretion was measured with the CTLL-2 assay as previouslydescribed. Results are presented in FIG. 6.

The human monoclonal antibody X3 was found to inhibit IL-2 secretion byEL4 cells induced by PFA-fixed monocytes, whether or not these werestimulated with LPS. A similar inhibition was obtained with the rabbitanti-IL-1α antibody but not with the rabbit anti-IL-1β antiserum. Theseresults indicate that the HuMAb X3 recognizes and neutralizes humanmembrane IL-1α.

Inhibition of IL-6 Production in Cocultures of Synoviocytes andMonocytes

To test the effect of HuMAb X3 on the production of IL-6 by thecoculture of synoviocytes and monocytes, elutriated blood monocytes werecultured for 24 hours with or without LPS (1 μg/ml) and then fixed ornot with PFA as described above. Serial dilutions of monocytepreparations were then incubated for 30 minutes at 37° C. with orwithout 1 μg/ml of HuMAb X3, 1 μg/ml non-related human IgG₄κ antibody or100 ng/ml of IL-1Ra. Cultures were performed in triplicate inflat-bottomed 96-well culture plates. Then 100 μl of synoviocytesuspension (5×10⁴ cells/ml) were added to each well. After 48 hours ofincubation, IL-6 secretion was measured in supernatants with a specificELISA. Controls performed without synoviocytes showed that, in contrastto unfixed monocytes, PFA-fixed monocytes were unable to secrete IL-6.The results are shown in FIG. 7.

The rheumatoid synovial tissue is composed of about 20%monocyte/macrophage/dendritic cells, 20% fibroblast-like cells(synoviocytes) and 30-50% T cells. This inflammatory tissue produces invivo and ex vivo high levels of proinflammatory cytokines, includingIL-6, TNF-α and IL-1β; see Miossec et al., Arthritis Rheum., 35:874-883(1992). A coculture of freshly isolated monocytes with synoviocytes fromlong term cultures results in the production of large amounts of IL-6but not of IL-10, IL-1β or TNF-α. Furthermore, a coculture ofsynoviocytes with PFA-fixed monocytes (unable to secrete IL-6) alsoproduced large amounts of IL-6. This indicates that IL-6 is most likelyproduced by synoviocytes following contact with monocytes.

As shown in FIG. 7, HuMAb X3 was found to strongly inhibit theproduction of IL-6 by coculture of synoviocytes with non-activatedmonocytes or LPS-stimulated monocytes (without or with PFA fixation).This finding is in accordance with the inhibitory effect of IL-1Ra.Thus, HuMAb X3 is able to interrupt an interaction between monocytes andsynoviocytes, and that interaction may represent a critical step in thedevelopment of rheumatoid inflammation.

Sequencing of the Variable Region Genes of the Anti-IL-1α HuMAb X3

RNA from the B cell clone X3 has been isolated according to thesingle-step method described by Chomczynski et al., Anal. Biochem.,162:156-159 (1987). Briefly, about 10⁷ cells from this clone were lysedin guanidinium thiocyanate denaturing solution. After acidification ofthe mixture with 2M sodium acetate, pH4, RNA was extracted with phenoland chloroform/isopentyl alcohol (24:1). RNA was then precipitated withisopropanol, the RNA pellet was redissolved in denaturing solution,reprecipitated with isopropanol, and washed with 75% ethanol.

cDNA was obtained by reverse transcription, using the SuperscriptReverse Transcriptase Kit (cat. 20898 BRL, Gaithersburg, Md., USA), witholigo dT₁₂₋₁₈ primers (Cat. 27.7858-01, Pharmacia, Uppsala, Sweden). ThecDNA was then used as template in the PCR. PCR amplifications wereperformed with Taq polymerase (Perkin Elmer, Norwalk, Conn.) using thereaction buffer provided by the manufacturers: Taq buffer: 1.5 mM MgCl₂,50 mM KCl, 10 mM Tris-HCl, pH 8.3 and 0.001% (w/v) gelatin. All PCRmixtures contained 200 ng of each primer, and 2.5 of Taq Polymerase.Amplifications were performed in a Trio-Thermoblock Thermal cycler(Biometra, GmbH) and consisted of 35 cycles of 1 minute denaturation at94° C., 2 min of primer annealing at 60° C., and 3 minutes extension at72° C. After the last cycle, the reaction mixtures were incubated for 10minutes at 72° C. to insure complete extension of all products. Theprimers were designed to include restriction sites, to allow for thedirectional cloning of the PCR products. For the heavy chain, primers(listed in SEQ ID NOS. 3, 4, 5, 6, 7 and 8) specific for the leadersequence of the six different human V_(H) families were usedindividually in conjunction with a primer (listed in SEQ ID NO. 9)located at the 3'-end of the gamma-constant region corresponding to thesub-class previously determined by isotyping the HuMAb by ELISA (IgG4).The light chain was amplified with individual combinations of primerscorresponding to the 3'-end of the kappa or lambda chain (listed in SEQID NOS. 10 and 11, respectively) in conjunction with a series of primers(listed in SEQ ID NOS. 12, 13, 14, 15, 16 and 17) annealing to theleader sequence of the different V kappa gene families or with a seriesof primers (listed in SEQ ID NOS. 18, 19, 20, 21 and 22) annealing tothe leader sequence of the different V lambda gene families. Thus,full-length heavy and light chains starting at the initiation codon inthe leader sequence and ending at the stop codon have been generated.Two independent PCRs were performed for both the heavy and the lightchains.

Those PCR products were loaded on agarose gels, and purified with GELase(Epicentre, cat. G21223, WI) according to the manufacturer'sinstructions. Purified PCR products from heavy and light chains wereused as template for sequencing reaction with leader PCR primers andwith primer hybridizing at the 5'-end of the gamma and of the kappa orlambda constant-region gene respectively. The sequencing reaction wasperformed on a 373 DNA Sequencer with TaqDyeDeoxy Terminator CycleSequencing Kit (both from Applied Biosystems Inc. Foster City, Calif.).Direct sequencing of both strands of the products of two independentPCRs were therefore obtained and compared. No difference was foundbetween the sequences of the two PCRs from the same cells. The sequenceof the V_(H) gene is listed in SEQ ID NO. 1 and the sequence of theV_(L) gene is listed in SEQ ID NO. 2 below. Framework (FR) andcomplementarity-determining regions (CDR) are as identified below, inagreement with the system of Kabat et al. (Kabat, E. A., T. T. Wu, H. M.Perry, K. S. Gottesman and C. Foeller (1987), Sequences of Proteins ofImmunological Interest, United States Department of Health and HumanServices, Bethesda, Md., p. 1).

    ______________________________________    amino acid residue nos.                          region    ______________________________________    V.sub.H Segment - SEQ ID NO. 1    -1 to -18             Signal peptide    +1 to +30             FR 1    +31 to +35            CDR 1    +36 to +49            FR 2    +50 to +66            CDR 2    +67 to 98             FR 3    +99 to +110           CDR 3    +111 to +122          JH 1    V.sub.L Segment - SEQ ID NO. 2    -1 to -22             Signal peptide    +1 to +23             FR 1    +24 to +34            CDR 1    +35 to +49            FR 2    +50 to +56            CDR 2    +57 to 88             FR 3    +89 to +95            CDR 3    +96 to +108           JK 4    ______________________________________

Sequences were compared with the Gene Bank release 77 using the DNAstar(WI, USA) software:

For the heavy-chain sequence (SEQ ID NO. 1), the most homologoussequence encoded was found to be HUMIGHYMG, a human V_(H) 3 germine geneOlee et al., J. Clin. Invest., 88:193-203 (1991)!. However, the level ofhomology was only 91.6% (25 nucleotide mismatches out of 300nucleotides), suggesting that the germline counterpart of HuMAb X3 heavychain is probably different from HUMIGHYAAG. It is therefore currentlyimpossible to assign mutation to any of the mismatches observed in theV_(H) segment. D segments are not unambiguously identifiable. There isone replacement mutation in the JH1 segment (120 Val/Phe).

For the light-chain sequence (SEQ ID NO. 2), the most homologoussequence encoded was found to be HUMIGKVJ2, a human Vkl germline genePech et al., J. Mol. Biol., 176:189-204 (1984)!. The level of homologyreaches 94.7% (14 nucleotide mismatches out of 264 nucleotides) stronglysuggesting that the germline counterpart of HuMAb X3 light chain isindeed HUMIGKVJ2. Analysis of somatic mutations in the V_(L) segmentshowed the following: the ratio of replacement mutations (R) vs. silentmutations (S) is R/S=5/1 in the CDRs, while R/S is 1/7 in the frameworksegments (FRs). D segments are not unambiguously identifiable. There arethree replacement mutations and one silent mutation in the JK4. Inconclusion, the kappa light chain of HuMAb X3 is heavily mutated, andthe ratio of R/S mutations in the CDRs vs FRs suggests strong selectionby the antigen.

Expression of Recombinant Anti-IL-1α HuMAb In Baculovirus

RNA from the B cell clone X3A-16G5 was isolated by the guanidiniumthiocyanate single-step method described by Chomczynski et al., supra.cDNA was obtained by reverse transcription of the RNA using aSuperscript Reverse Transcriptase Kit with oligo dT₁₂₋₁₈ primers (Cat.27.7858-01, Pharmacia, Uppsala, Sweden). The cDNA was then used as atemplate in the PCR performed with Taq polymerase. The primers weredesigned to include EcoR1 and Not1 restriction sites, to allow for thedirectional cloning of the PCR products into the baculovirus vectorpVL-1393. For the heavy chain, primer (listed in SEQ ID NO. 5) specificfor the V_(H) 3 leader sequence of the human V_(H) family was used inconjunction with a primer (listed in SEQ ID NO. 9) located at the 3'-endof the gamma constant region corresponding to the sub-class previouslydetermined by isotyping the HuMAb by ELISA (IgG₄). The light chain wasamplified with a primer (listed in SEQ ID NO. 10) corresponding to the3'-end of the kappa chain in conjunction with a primer (listed in SEQ IDNO. 12) annealing to the leader sequence of the V kappa 1 gene family.Thus, full-length heavy and light chains cDNAs were generated. Twoindependent PCRs were performed for both the heavy and the light chains.

After appropriate restriction cleavage of these PCR products, bothfull-length heavy and light chains were cloned in baculovirus vectorrestricted with the same enzymes. Heavy and light chains were clonedindividually in distinct pVL1393 baculovirus vectors (Invitrogen Co, SanDiego, Calif.). The recombinant vectors were transfected in competentDH5αE. coli bacteria (Gibco BRL, Gaithersburg, Md.), and 10 singlecolonies were selected. 100 ml culture of each bacterial clone wereobtained, and vector DNA was purified with Qiagen plasmid-Kit (Diagen,GmbH). Both strands of the complete insert from double-stranded DNAvector were sequenced with (1) two primers flanking the insert--thefirst (listed in SEQ ID NO. 23) annealing 5' in the promoter region ofthe polyhedrin gene and the second (listed in SEQ ID NO. 24) annealing3' in the polyhedrin gene itself; and (2) a series of forward primersand backward primers distributed about 400 bp apart along the heavy- andthe light-chain sequences; i.e., the forward primers for the heavy chainare listed in SEQ ID NOS. 25 and 26, the backward primers for the heavychain are listed in SEQ ID NOS. 27, 28 and 29, and the backward primerfor the light chain is listed in SEQ ID NO. 30. Double-stranded DNAsequencing was done on a 373 DNA Sequencer with TaqDyeDeoxy TerminatorCycle Sequencing Kit (both from Applied Biosystems Inc., Foster City,Calif.). A recombinant baculovirus vector clone was selected for boththe heavy and the light chains, which showed perfect match with thevariable-region sequences obtained from the PCR products, and with thepublished sequences of the constant regions of the heavy gamma 4 andkappa light chains respectively. Recombinant baculovirus vectors werecotransfected with wild type baculovirus DNA in Sf9 insect cells, usingthe transfection module (Invitrogen Co, San Diego, Calif.). Recombinantbaculoviruses recovered from the cell culture supernatant of thesetransfected cells were then cloned in Sf9 cells by limiting dilution andscreened by hybridization with the labeled inserts. After two runs ofcloning, followed by production, recombinant baculoviruses containingthe heavy- or the light-chain cDNAs were titrated, and used to infectinsect cells at a Multiplicity of Infection (MOI) of 5. After 5 days ofculture, production of human heavy or light chain was confirmed by ELISAand/or by in vivo labeling. One baculovirus clone expressing the heavychain and one expressing the light chain were used to co-infect Sf9cells, each at a MOI of 5. After 5 days of infection, the presence inthe supernatant of an antibody binding specifically to human IL-1α wasconfirmed by immunoprecipitation.

Supernatants of insect cells infected with (1) recombinant baculoviruscontaining cDNA of the light chain of X3, (2) with baculoviruscontaining cDNA of X3 heavy chain, (3) with baculoviruses containingcDNAs of both the X3 heavy and the X3 light chains and (4) withbaculoviruses containing cDNAs of both heavy and light chains of anon-related human IgG antibody, were assayed in the StandardImmunoprecipitation Protocol with Protein G described above. The resultsare shown in FIG. 8A. The results demonstrate that supernatantscontaining the X3 heavy and light chains precipitated ¹²⁵ I-IL-1α, whilesupernatants containing X3 light chain alone, X3 heavy chain alone, orthe heavy and light chains of the non-related human IgG did notimmunoprecipitate ¹²⁵ I-IL-1α.

Furthermore, the natural HuMAb X3 and the recombinant form of X3 wereemployed in the assay "Cross-Reactivity of the Human Monoclonal AntibodyX3" described above. The results are shown in FIG. 8B, which demonstratethat, as with the natural HuMAb X3, the recombinant form of X3specifically recognized human IL-1α, since the ¹²⁵ I-IL-1αimmunoprecipitation was not protected by preincubation of X3 with anexcess of unlabeled human IL-1β and IL-1Ra.

This recombinant form of X3 was purified from Sf9 cell conditionedmedium on a protein G column as previously described for thepurification of natural HuMAb X3. The purified recombinant X3 wasassayed in the assay "Affinity for Human IL-1α" described above. Theobtained equilibrium affinity constant (K_(a)) value was 1.4×10¹⁰ M⁻¹.Finally, the purified recombinant X3 was assayed in the assays"Inhibition of Human IL-1α-induced IL-2 Secretion by EL4 cells" and"Inhibition of Human IL-1α-induced IL-6 production by HumanSynoviocytes" described above. Results shown in FIG. 9A and FIG. 9Bindicate that the recombinant HuMAb X3 inhibited human IL-1α biologicalactivity with the same efficiency as the natural HuMAb X3.

Repertoire Cloning

As demonstrated above, by dilution cloning of an immortalized and/oractivated B cell population in accordance with the present invention, aseries of amplified B cell subpopulations can be provided for screeningfor antibodies that bind to the desired antigen, e.g., by the standardand derived immunoprecipitation protocols described above. Thus, byusing the immortalization, amplification and screening techniques of thepresent invention, it is possible to produce and identify animmortalized and/or secreting B cell subpopulation that consists of fromabout 5 to about 50 different, amplified B cell clones, at least one ofwhich expresses a HuMAb against the desired antigen, e.g. a humancytokine such as IL-1β, TNF-α, IL-6, IL-10 etc.

For example, as discussed above, we produced amplified, immortalizedand/or secreting B cell subpopulations from 15 patients expressingHuMAbs against human IL-1α. Each of these subpopulations contained fromabout 5 to about 50 different amplified B cell clones in a total numberof about 5×10⁵ to about 50×10⁵ B cells.

Thus, the percentage of B cells producing HuMAbs against the antigen ofinterest in a B cell subpopulation of the present invention is greatlyenhanced in comparison to other techniques which start with naturallyoccurring B cell populations. This amplified subpopulation can make itpossible to uncover HuMAbs to human cytokines even when isolating asingle clone, as we accomplished with the X3 clone discussed above, isnot possible.

Starting with an amplified, immortalized and/or secreting B cellsubpopulation in accordance with the present invention, including, forexample, 40 different B cell clones, the number of possible V_(H) /V_(L)combinations from a cDNA library encoding the V_(H) segments and V_(L)segments from these B cells is 1600 (40×40). This very low number makesit far easier to isolate the specific combination of V_(H) and V_(L)segments responsible for the one (or more) amplified HuMAb clone in thesubpopulation which binds to the desired antigen. Thus, by applyingstandard techniques such as repertoire cloning and phage display to theamplified, immortalized and/or secreting B cell subpopulation of theinvention, a series of HuMAbs against the desired antigen can beidentified and isolated by recombinant techniques.

For example, a cDNA library encoding the mRNA repertoire of V_(H) and/orV_(L) segments of all the HuMAbs expressed in such a subpopulation(e.g., a subpopulation screened as containing a clone expressing a HuMAbto IL-10) can be prepared by PCR amplification of the mRNA from thesubpopulation using appropriate primers. These repertoire cloningtechniques are now standard in the art; see, for example, Marks et al.,J. Mol. Biol., 222:581-597 (1991); Huse et al., Science, 246:1275-1281(1989); WO 90/14430; WO 92/15678; WO 91/16427; and WO 92/01047. The DNAsencoding the V_(H) and V_(L) segments may be assembled into appropriatevectors for direct cloning and expression in a host, e.g., by themethods described in Hoogenboom et al., Nucleic Acids Research,19:4133-4137 (1991). The expressed V_(H) and V_(L) segments or Fab(e.g., for an anti-IL-10 HuMAb) may then be screened for binding to thedesired antigen by the standard and derived immunoprecipitationprotocols described above using labeled antigen, e.g. ¹²⁵ I-IL-10. TheDNA encoding the V_(H) or V_(L) segments from the identified clones canthen be sequenced and operatively linked to DNA segments encoding theconstant regions for the desired HuMAb isotype heavy or light chains,e.g., heavy chains or κ or λ light chains of IgG₁, IgG₂, IgG₃, IgG₄,IgA, etc., to create a complete HuMAb or a fragment thereof (e.g., Fab,F(ab')₂, Fv etc.) against the desired antigen, e.g., human IL-10.

Alternatively, the cDNA repertoire encoding the V_(H) and/or V_(L)segments can be included in a vector appropriate to display the V_(H)and/or V_(L) segments on the surface of a suitable host. See, forexample, Marks et al., J. Mol. Biol., 222:581-597 (1991) and Hoogenboomet al., supra, which disclose methodologies for displaying Fv, scFv orFab fragments of such a cDNA library on the surface of bacteriophage.The host cells (e.g., phage) displaying the scFv on their surface thatbind to the desired antigen can then be identified by ELISA or any othersuitable assay. The DNA encoding the V_(H) and/or V_(L) segments frombinding host cells can be separated and reassembled into appropriatevectors for direct cloning and expression in a host, e.g., by the methoddescribed in the Marks et al. article. The DNA sequences can then beassembled into a full-length HuMAb) or fragment thereof by themethodologies described above.

An amplified B-cell subpopulation of the present invention as describedabove may be employed with single cell and multiple cell PCR techniquesto yield the DNA sequences encoding the variable regions of the HuMAbsproduced by these cells. For example, a CD40-crosslinked B cellpopulation of the invention (which may also be EBV-transformed) may bediluted to provide either a small number of B cells (e.g., 10 B cellsper well) or (on average) a single B cell or less per well. Themethodologies disclosed, for example, in Larrick et al., Biotechnology,7:934 (1989), Embleton et al., Nucleic Acids Research, 10:3831-3837(1992), Liu et al., Proc. Natl. Acad. Sci., 89: 7610-7614 (1992), andLew et al. Immunology, 75:3-9 (1992), may be employed to obtain accurateand complete heavy- and light-chain variable region genes (V_(H) andV_(L) genes) from these cells. These DNA sequences may be included in anappropriate recombinant system to express, for example, an Fv or scFv,and the fact that they represent a HuMAb to the desired antigen may thenbe confirmed by the immunoprecipitation assays described above forbinding to the desired antigen. Previous isotyping of the active HuMAbsin the B-cell starting population or subpopulation may then be used toconstruct the full-length HuMAb.

The activity of the HuMAb may then be confirmed by conventional in vitroand in vivo biological assays. For example, for TNF-α, IL-1β and IL-6,the assays and models reviewed in Dinarello, Eur. Cytokine Netw., 3:7-17(1992) may be employed. For human IL-10 the cytokine synthesisinhibitory factor (CSIF) assay described in Florentino et al., J. Exp.Med., 170:2081-2095 (1989) or the property of IL-10 to induceproliferation and Ig secretion by human B cellar as described in Roussetet al., Proc. Natl. Acad. Sci. USA, 89:1890-1893 (1992) may be employed.

It is most likely that the pairs of heavy and light chains identifiedunder the above conditions will be those of the identified antibody,since the screening procedures described above are selective for highaffinity antibody which can only be obtained with a given combination ofheavy and light chain. Furthermore, the identification of the heavy- andlight-chain isotypes in the supernatants of the oligoclonal cell lineswill also be of great help to determine whether the selected pair doesindeed correspond to the initially identified clone.

While the present invention has been described in conjunction with thespecific embodiments set forth above, many alternatives, modificationsand variations thereof will be apparent to those of ordinary skill inthe art. All such alternatives, modifications and variations fall withinthe spirit and scope of the present invention.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES:   30    - (2) INFORMATION FOR SEQ ID NO: 1:    -      (i) SEQUENCE CHARACTERISTICS:    #base pairsA) LENGTH:   423    #acid     (B) TYPE:   nucleic    #double   (C) STRANDEDNESS:              (D) TOPOLOGY:   line - #ar    -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 1:    - ATG GAG TTT GGG CTG AGC TGG GTT TTC CTC GT - #T GCT CTT TTA AGA GGT      48    Met Glu Phe Gly Leu Ser Trp Val Phe Leu Va - #l Ala Leu Leu Arg Gly    - GTC CAG TGT CAG GTG CAA CTG GTG GAA TCT GG - #G GGA GGC GTG GTC CAG      96    Val Gln Cys Gln Val Gln Leu Val Glu Ser Gl - #y Gly Gly Val Val Gln    #          10    - CCT GGG AGG TCC CTG AGA CTC TCC TGT ACA GC - #C TCT GGA TTC ACC TTC     144    Pro Gly Arg Ser Leu Arg Leu Ser Cys Thr Al - #a Ser Gly Phe Thr Phe    #    25    - AGT ATG TTT GGT GTC CAC TGG GTC CGC CAG GC - #C CCA GGC AAG GGG CTG     192    Ser Met Phe Gly Val His Trp Val Arg Gln Al - #a Pro Gly Lys Gly Leu    #45    - GAG TGG GTG GCA GCT GTG TCA TAT GAT GGA AG - #C AAT AAG TAC TAT GCA     240    Glu Trp Val Ala Ala Val Ser Tyr Asp Gly Se - #r Asn Lys Tyr Tyr Ala    #                60    - GAG TCC GTG AAG GGC CGA TTC ACC ATC TCC AG - #A GAC AAT TCC AAG AAC     288    Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Ar - #g Asp Asn Ser Lys Asn    #            75    - ATC TTA TTT CTA CAA ATG GAC AGC CTG AGA CT - #T GAG GAC ACG GCT GTC     336    Ile Leu Phe Leu Gln Met Asp Ser Leu Arg Le - #u Glu Asp Thr Ala Val    #        90    - TAT TAC TGT GCT AGA GGC CGG CCC AAG GTC GT - #A ATA CCA GCA CCT TTG     384    Tyr Tyr Cys Ala Arg Gly Arg Pro Lys Val Va - #l Ile Pro Ala Pro Leu    #    105    #    423C TGG GGC CAG GGA ACC CTG GTC ACC TT - #C TCC TCA    Ala His Trp Gly Gln Gly Thr Leu Val Thr Ph - #e Ser Ser    110                 1 - #15                 1 - #20    - (2) INFORMATION FOR SEQ ID NO: 2:    -      (i) SEQUENCE CHARACTERISTICS:    #base pairsA) LENGTH:   390    #acid     (B) TYPE:   nucleic    #double   (C) STRANDEDNESS:              (D) TOPOLOGY:   line - #ar    -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 2:    - ATG GAC ATG AGG GTC CCC GCT CAG CTC CTG GG - #G CTC CTG CTG CTC TGG      48    Met Asp Met Arg Val Pro Ala Gln Leu Leu Gl - #y Leu Leu Leu Leu Trp    10    - TTC CCA GGT TCC AGA TGC GAC ATC CAG ATG AC - #C CAG TCT CCA TCT TCC      96    Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Th - #r Gln Ser Pro Ser Ser    #  10    - GTG TCT GCA TCT GTA GGA GAC AGA GTC ACC AT - #C ACT TGT CGG GCG AGT     144    Val Ser Ala Ser Val Gly Asp Arg Val Thr Il - #e Thr Cys Arg Ala Ser    #                25    - CAG GGT ATT AGC AGT TGG TTA GCC TGG TAT CA - #G CAG AAA CCA GGA AAG     192    Gln Gly Ile Ser Ser Trp Leu Ala Trp Tyr Gl - #n Gln Lys Pro Gly Lys    #            40    - GCC CCG AAG CTC TTG ATC TAT GAA GCA TCC AA - #T TTG GAA ACT GGG GTC     240    Ala Pro Lys Leu Leu Ile Tyr Glu Ala Ser As - #n Leu Glu Thr Gly Val    #        55    - CCA TCA AGA TTC AGC GGC AGT GGA TCT GGG TC - #A GAT TTC ACC CTC ACC     288    Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Se - #r Asp Phe Thr Leu Thr    #    70    - ATC AGC AGC CTG CAG CCT GAA GAT TTT GCA AC - #T TAT TAT TGT CAA CAG     336    Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Th - #r Tyr Tyr Cys Gln Gln    #90    - ACT AGC AGT TTT CTC CTC AGT TTC GGC GGC GG - #G ACC AAG GTG GAG CAC     384    Thr Ser Ser Phe Leu Leu Ser Phe Gly Gly Gl - #y Thr Lys Val Glu His    #                105    #          390    Lys Arg    - (2) INFORMATION FOR SEQ ID NO: 3:    -      (i) SEQUENCE CHARACTERISTICS:    #base pairsA) LENGTH:   27    #acid     (B) TYPE:   nucleic    #single   (C) STRANDEDNESS:              (D) TOPOLOGY:   line - #ar    -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 3:    #             27   GGAC CTGGAGG    - (2) INFORMATION FOR SEQ ID NO: 4:    -      (i) SEQUENCE CHARACTERISTICS:    #base pairsA) LENGTH:   29    #acid     (B) TYPE:   nucleic    #single   (C) STRANDEDNESS:              (D) TOPOLOGY:   line - #ar    -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 4:    #            29    TACT TTGTACCAC    - (2) INFORMATION FOR SEQ ID NO: 5:    -      (i) SEQUENCE CHARACTERISTICS:    #base pairsA) LENGTH:   27    #acid     (B) TYPE:   nucleic    #single   (C) STRANDEDNESS:              (D) TOPOLOGY:   line - #ar    -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 5:    #             27   TTGG GCTGAGC    - (2) INFORMATION FOR SEQ ID NO: 6:    -      (i) SEQUENCE CHARACTERISTICS:    #base pairsA) LENGTH:   29    #acid     (B) TYPE:   nucleic    #single   (C) STRANDEDNESS:              (D) TOPOLOGY:   line - #ar    -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 6:    #            29    ACCT GTGGTTCTT    - (2) INFORMATION FOR SEQ ID NO: 7:    -      (i) SEQUENCE CHARACTERISTICS:    #base pairsA) LENGTH:   29    #acid     (B) TYPE:   nucleic    #single   (C) STRANDEDNESS:              (D) TOPOLOGY:   line - #ar    -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 7:    #            29    CAAC CGCCATCCT    - (2) INFORMATION FOR SEQ ID NO: 8:    -      (i) SEQUENCE CHARACTERISTICS:    #base pairsA) LENGTH:   29    #acid     (B) TYPE:   nucleic    #single   (C) STRANDEDNESS:              (D) TOPOLOGY:   line - #ar    -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 8:    #            29    TCTC CTTCCTCAT    - (2) INFORMATION FOR SEQ ID NO: 9:    -      (i) SEQUENCE CHARACTERISTICS:    #base pairsA) LENGTH:   31    #acid     (B) TYPE:   nucleic    #single   (C) STRANDEDNESS:              (D) TOPOLOGY:   line - #ar    -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 9:    #          31      TCAT TTACCCGGAG A    - (2) INFORMATION FOR SEQ ID NO: 10:    -      (i) SEQUENCE CHARACTERISTICS:    #base pairsA) LENGTH:   34    #acid     (B) TYPE:   nucleic    #single   (C) STRANDEDNESS:              (D) TOPOLOGY:   line - #ar    -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 10:    #        34        ACAC TCTCCCCTGT TGAA    - (2) INFORMATION FOR SEQ ID NO: 11:    -      (i) SEQUENCE CHARACTERISTICS:    #base pairsA) LENGTH:   38    #acid     (B) TYPE:   nucleic    #single   (C) STRANDEDNESS:              (D) TOPOLOGY:   line - #ar    -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 11:    #     38           ATGA ACATTCTGTA GGGGCCAC    - (2) INFORMATION FOR SEQ ID NO: 12:    -      (i) SEQUENCE CHARACTERISTICS:    #base pairsA) LENGTH:   36    #acid     (B) TYPE:   nucleic    #single   (C) STRANDEDNESS:              (D) TOPOLOGY:   line - #ar    -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 12:    #       36         TGAG GGTCCCCGCT CAGCTC    - (2) INFORMATION FOR SEQ ID NO: 13:    -      (i) SEQUENCE CHARACTERISTICS:    #base pairsA) LENGTH:   33    #acid     (B) TYPE:   nucleic    #single   (C) STRANDEDNESS:              (D) TOPOLOGY:   line - #ar    -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 13:    #         33       CGAG GGCCCCCACT CAG    - (2) INFORMATION FOR SEQ ID NO: 14:    -      (i) SEQUENCE CHARACTERISTICS:    #base pairsA) LENGTH:   29    #acid     (B) TYPE:   nucleic    #single   (C) STRANDEDNESS:              (D) TOPOLOGY:   line - #ar    -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 14:    #            29    TGCA GACCCAGGT    - (2) INFORMATION FOR SEQ ID NO: 15:    -      (i) SEQUENCE CHARACTERISTICS:    #base pairsA) LENGTH:   33    #acid     (B) TYPE:   nucleic    #single   (C) STRANDEDNESS:              (D) TOPOLOGY:   line - #ar    -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 15:    #         33       TCCC TGCTCAGCTC CTG    - (2) INFORMATION FOR SEQ ID NO: 16:    -      (i) SEQUENCE CHARACTERISTICS:    #base pairsA) LENGTH:   29    #acid     (B) TYPE:   nucleic    #single   (C) STRANDEDNESS:              (D) TOPOLOGY:   line - #ar    -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 16:    #            29    CCCC AGCGCAGCT    - (2) INFORMATION FOR SEQ ID NO: 17:    -      (i) SEQUENCE CHARACTERISTICS:    #base pairsA) LENGTH:   30    #acid     (B) TYPE:   nucleic    #single   (C) STRANDEDNESS:              (D) TOPOLOGY:   line - #ar    -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 17:    #           30     TCCCA  GGTTCACCTC    - (2) INFORMATION FOR SEQ ID NO: 18:    -      (i) SEQUENCE CHARACTERISTICS:    #base pairsA) LENGTH:   29    #acid     (B) TYPE:   nucleic    #single   (C) STRANDEDNESS:              (D) TOPOLOGY:   line - #ar    -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 18:    #            29    GCTC CCCTCTCCT    - (2) INFORMATION FOR SEQ ID NO: 19:    -      (i) SEQUENCE CHARACTERISTICS:    #base pairsA) LENGTH:   33    #acid     (B) TYPE:   nucleic    #single   (C) STRANDEDNESS:              (D) TOPOLOGY:   line - #ar    -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 19:    #         33       GGAC TCCTCTCTTT CTG    - (2) INFORMATION FOR SEQ ID NO: 20:    -      (i) SEQUENCE CHARACTERISTICS:    #base pairsA) LENGTH:   29    #acid     (B) TYPE:   nucleic    #single   (C) STRANDEDNESS:              (D) TOPOLOGY:   line - #ar    -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 20:    #            29    GGGC TCCACTACT    - (2) INFORMATION FOR SEQ ID NO: 21:    -      (i) SEQUENCE CHARACTERISTICS:    #base pairsA) LENGTH:   29    #acid     (B) TYPE:   nucleic    #single   (C) STRANDEDNESS:              (D) TOPOLOGY:   line - #ar    -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 21:    #            29    GGAT CCCTCTCTT    - (2) INFORMATION FOR SEQ ID NO: 22:    -      (i) SEQUENCE CHARACTERISTICS:    #base pairsA) LENGTH:   30    #acid     (B) TYPE:   nucleic    #single   (C) STRANDEDNESS:              (D) TOPOLOGY:   line - #ar    -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 22:    #           30     GGGC TCTGCTGCTC    - (2) INFORMATION FOR SEQ ID NO: 23:    -      (i) SEQUENCE CHARACTERISTICS:    #base pairsA) LENGTH:   25    #acid     (B) TYPE:   nucleic    #single   (C) STRANDEDNESS:              (D) TOPOLOGY:   line - #ar    -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 23:    #               25 GATT ATTCA    - (2) INFORMATION FOR SEQ ID NO: 24:    -      (i) SEQUENCE CHARACTERISTICS:    #base pairsA) LENGTH:   17    #acid     (B) TYPE:   nucleic    #single   (C) STRANDEDNESS:              (D) TOPOLOGY:   line - #ar    -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 24:    #   17             G    - (2) INFORMATION FOR SEQ ID NO: 25:    -      (i) SEQUENCE CHARACTERISTICS:    #base pairsA) LENGTH:   21    #acid     (B) TYPE:   nucleic    #single   (C) STRANDEDNESS:              (D) TOPOLOGY:   line - #ar    -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 25:    #21                CTCC C    - (2) INFORMATION FOR SEQ ID NO: 26:    -      (i) SEQUENCE CHARACTERISTICS:    #base pairsA) LENGTH:   27    #acid     (B) TYPE:   nucleic    #single   (C) STRANDEDNESS:              (D) TOPOLOGY:   line - #ar    -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 26:    #             27   CCAA GGTGGAC    - (2) INFORMATION FOR SEQ ID NO: 27:    -      (i) SEQUENCE CHARACTERISTICS:    #base pairsA) LENGTH:   26    #acid     (B) TYPE:   nucleic    #single   (C) STRANDEDNESS:              (D) TOPOLOGY:   line - #ar    -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 27:    #              26  CTGA CGGTCC    - (2) INFORMATION FOR SEQ ID NO: 28:    -      (i) SEQUENCE CHARACTERISTICS:    #base pairsA) LENGTH:   28    #acid     (B) TYPE:   nucleic    #single   (C) STRANDEDNESS:              (D) TOPOLOGY:   line - #ar    -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 28:    #             28   TTCT CGGGGCTG    - (2) INFORMATION FOR SEQ ID NO: 29:    -      (i) SEQUENCE CHARACTERISTICS:    #base pairsA) LENGTH:   21    #acid     (B) TYPE:   nucleic    #single   (C) STRANDEDNESS:              (D) TOPOLOGY:   line - #ar    -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 29:    #21                GTGC C    - (2) INFORMATION FOR SEQ ID NO: 30:    -      (i) SEQUENCE CHARACTERISTICS:    #base pairsA) LENGTH:   20    #acid     (B) TYPE:   nucleic    #single   (C) STRANDEDNESS:              (D) TOPOLOGY:   line - #ar    -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 30:    # 20               ACAG    __________________________________________________________________________

We claim:
 1. A human monoclonal antibody against a human IL-1α or ahuman IL-1α binding fragment of said antibody characterized in that itcomprises a complementary determining region of an amino acid sequencedefined by amino acids 1-122 of an amino acid sequence encoded by thenucleic acid sequence shown in SEQ ID NO:1 or by a complementarydetermining region somatic variant thereof and/or of an amino acidsequence defined by amino acids 1-108 of an amino acid sequence encodedby the nucleic acid sequence shown in SEQ ID NO:2; or by a complementarydetermining region somatic variant thereof.
 2. A human monoclonalantibody against a human IL-1α or a human IL-1α binding fragment of saidantibody characterized in that it comprises a V_(H) segment having anamino acid sequence defined by amino acids 1-122 of an amino acidsequence encoded by the nucleic acid sequence shown in SEQ ID NO:1 or bya complementary determining region somatic variant thereof, and/or aV_(L) segment having an amino acid sequence defined by amino acids 1-108of an amino acid sequence encoded by the nucleic acid sequence shown inSEQ ID NO:2 or by a complementary determining region somatic variantthereof.
 3. The antibody or fragment according to claim 2, characterizedin that it comprises a V_(H) segment having an amino acid sequencedefined by amino acids 1-122 of an amino acid sequence encoded by thenucleic acid sequence shown in SEQ ID NO:1 and/or a V_(L) segment havingan amino acid sequence defined by amino acids 1-108 of an amino acidsequence encoded by the nucleic acid sequence shown in SEQ ID NO:2. 4.The antibody according to claim 2, characterized in that it comprisesV_(H) and V_(L) segments having the amino acid sequences defined byamino acids 1-122 of an amino acid sequence encoded by the nucleic acidsequence shown in SEQ ID NO:1 and amino acids 1-108 of an amino acidsequence encoded by the nucleic acid sequence shown in SEQ ID NO:2,respectively, or which comprises a complementary determining regionsomatic variant of one or both of said amino acid sequences.
 5. Theantibody according to claim 2, characterized in that it is the IgG₄isotype.
 6. The fragment according to claim 3, characterized in that itcomprises a Fv, single-chain Fv, Fab or F(ab')₂ fragment.
 7. An isolatednucleic acid characterized in that it comprises: a nucleotide sequencedefined by base numbers 58423 of SEQ ID NO:1 or by a complementarydetermining region encoding somatic variant thereof, and/or a nucleotidesequence defined by base numbers 67-390 of SEQ ID NO:2 or by acomplementary determining region encoding somatic variant thereof.
 8. Anisolated nucleic acid according to claim 7, characterized in that itcomprises a nucleotide sequence defined by base numbers 58-423 of SEQ IDNO:1 and/or base numbers 67-390 of SEQ ID NO:2.